Microbiology Unit 3

Chemoorganotroph

Organisms that get their energy and electrons by oxidizing organic chemical compounds (e.g. sugars, fats, proteins)

Metabolism

Consists of catabolism and anabolism

Catabolism

• Precursor/metabolite generation

• Substrate —> catabolic pathway —> ATP + NAD(P)H + building blocks

• Leads to the production of 12 key precursors

• Reducing power (NADH + NADPH)

• ATP

Anabolism

• Assembly

• ATP + NAD(P)H + building blocks —> anabolic pathway —> C₁H_1.8O_0.5N_0.2 (make themselves)

Redox reactions

• Explains the movement of electrons from donor to acceptor

• NADH + H⁺ + ½ O₂ —> H₂O + NAD⁺ (E₀ = 1.14 V)

Standard Reduction Potential

• Ability to donate or accept electrons

• More -E₀ —> better electron donor

• More +E₀ —> better electron acceptor

Electron Bifurcation

• Couples an energetically favorable reaction to an unfavorable one

• Used to perform more niche reactions

Catabolic Diversity

• There are several ways to obtain carbon, electrons, energy, etc

• Not only from organic materials; can be from light, inorganic molecules, chemicals, CO₂

Photolithoautotroph

• Energy source = light

• Electron source = inorganic e^- donor

• Carbon source = CO₂

• e.g. purple and green sulfur bacteria, cyanobacteria, diatoms

Photoorganoheterotroph

• Energy source = light

• Electron source = organic e^- donor

• Carbon source = organic carbon

• e.g. purple nonsulfur bacteria, green nonsulfur bacteria

Chemolithoautotroph

• Energy source = inorganic chemicals

• Electron source = inorganic e^- donor

• Carbon source = CO₂

• e.g. sulfur-oxidizing bacteria, hydrogen-oxidizing bacteria, methanogens, nitrifying bacteria, iron-oxidizing bacteria

Chemolithoheterotroph

• Energy source = inorganic chemicals

• Electron source = inorganic e^- donor

• Carbon source = organic carbon

• e.g. some sulfur-oxidizing bacteria

Chemoorganoheterotroph

• Energy source = organic chemicals, often same as C source

• Electron source = organic e^- donor, often same as C source

• Carbon source = organic carbon

• e.g. most nonphotosynthetic microbes, including most pathogens, fungi, and many protists

• Incoming reduced organic carbon serves 3 purposes: energy, electrons, and C for anabolism

Glucose

• Excellent source of energy

• Can donate up to 24 e^-

Pathways to convert glucose —> PGALD to produce pyruvate

1. Glycolysis (Embden-Meyerhof-Parnas Pathway)

2. Pentose Phosphate Pathway (PPP)

3. Entner-Doudoroff Pathway (ED)

Glycolysis

• 2 key stages

  • 6C Phase: Glucose (C₆) —> split into 2 PGALD (C₃); must invest 2 ATP

  • 3C Phase: Oxidation of PGALD —> pyruvate

• Substrate level phosphorylation

• Contributes to anabolic pathways

Substrate level phosphorylation

• Individually moving a phosphate from 1 molecule onto ADP

• Inefficient

Pentose Phosphate Pathway

• Can run parallel to glycolysis

• Breaks down G6P to generate reducing equivalents + anabolic precursors (5-carbon sugars which are needed for DNA)

• 3 key stages:

  • Step 1: oxidation-decarboxylation of G6P

  • Step 2: isomerization of ribulose

  • Step 3: sugar rearrangement reactions

• Contributes to anabolic pathways

  • Nucleic acid synthesis

  • Aromatic amino acid synthesis

• Some organisms rely solely on this

Entner-Doudoroff Pathway

• Alternative metabolic pathways to catabolize glucose —> pyruvate

  • Growth on aldonic acids aka sugar acids which can’t be used in glycolysis

  • Less enzyme requirements; advantageous in limited nutrient niches

• Generates only 1 ATP + 1 NADH + 1 NADPH

• Similar initial reactions EMP but more inefficient

• Conversion of gluconate to KDGP leads to formation of 1 pyruvate + 1 PGALD

• Only 1 ATP formed

• Specific to prokaryotes

• Some organisms rely solely on this

• Direct production of NADPH

Disaccharides

Broken apart by hydrolysis or phosphorylation reactions

TCA Cycle

• 1 Acetyl-CoA oxidized —> 2 CO₂ + 3 NADH + 1 FADH₂ + 1 GTP

  • Pyruvate oxidized —> acetyl-CoA in a process called pyruvate dehydrogenase

    • Pre-req to TCA cycle

    • Net production of 1 NADH

• Contributes to anabolic pathways (mostly produces reducing power)

ETC carriers

• Works similarly between bacterium and eukaryotes

• Quinones are the complexes embedded in the membrane

  • Differs from bacteria to bacteria (unlike eukarya which have the same set up every time)

Chemoorganotrophic fueling processes

• Use reduced organic compounds for catabolic and anabolic processes

• Processes used to catabolize energy source:

  • Respiration

  • Fermentation

Respiration

• Involves use of the ETC (most of the time)

• 2 types:

  • Aerobic - TEA is oxygen

  • Anaeorbic - TEA is different oxidized molecule e.g. NO₃^-, SO₄^2-, Fe³⁺

  • PMF is generated and used to synthesize ATP as e^- pass through the ETC to the TEA

Electron Transport Chain

• Electron carriers are membrane-bound

  • Bacteria don’t have a dedicated organelle for this

    • Gram +: PM

    • Gram -: IM

• 1st electron carrier has the most negative reduction potential and each successive carrier is slightly less negative

Reducing power

• NAD+ - nicotinamide adenine dinucleotide

  • Reduced to NADH

• NADP+ - nicotinamide adenine dinucleotide phosphate

  • Reduced to NADPH

• FAD - flavin adenine dinucleotide

  • Redox active cofactor

• FMN - flavin mononucleotide

  • Redox active cofactor

Electron carriers

• Coenzyme Q (CoQ) - ubiquinone

  • Soluble lipid carrier

  • Tail group embedded in membrane

  • Head harvest e^-

• Cytochromes

  • Usually involves intermediates

  • Contains heme groups (ring-shaped to hold iron)

• Ferredoxin & other non-heme iron proteins

  • Iron-sulfur cluster containing protein

Aerobic Respiration (eukaryotes)

NADH/FADH₂ —> Complex I or II —> CoQ —> Complex III —> Cyt c —> Complex IV —> O₂

Aerobic Respiration (bacteria)

• Located in PM

• Flexible - electron carriers can be replaced or different terminal oxidases may be used to respond to different environmental conditions

• Branched - electrons may enter the chain at several points and leave through several terminal oxidases

• >46 respiratory dehydrogenases to harvest e^-

Why does E. coli use two different cytochrome oxidases?

• Both utilize O₂ as a TEA

  • 1 is made/used when O₂ is at high levels

  • The other when O₂. is at low levels

• Different affinities based on nutrient availability

• Different efficiencies

Anaerobic Respiration

• Uses electron acceptors other than oxygen

• Generally yields less ATP than aerobic respiration because of a lower difference in standard reduction potentials

Denitrification

• Reduction of nitrate to nitrogen gas

• Important environmentally

Aerobic vs. Anaerobic Respiration

• Similarities -

  • Organic substrate is oxidized (CH₂O —> CO₂)

  • Resulting NADH and FADH₂ used to “kick off” the ETC

  • Still couples energetic favorability of moving electron to unfavorably pumping protons (PMF)

• Differences -

  • O₂ is not the TEA

    • SO₄, NO₃, Fe³⁺ are common TEA

    • Less energy is obtained because of lower difference between standard reduction potential (but as long as there is a difference they will use it)

Electron acceptors used in anaerobic respiration by E. coli + enteric bacteria

• Electron acceptor: NO₃^-

• Reduced products: NO₂^-

Electron acceptors used in anaerobic respiration by Paracoccus denitrificans + Pseudomonas

• Electron acceptor: NO₃^-

• Reduced products: NO₂^-, N₂O, N₂

Electron acceptors used in anaerobic respiration by methanogens

• Electron acceptor: CO₂

• Reduced products: CH₄

Electron acceptors used in anaerobic respiration by acetogens

• Electron acceptor: CO₂

• Reduced products: acetate

Electron acceptors used in anaerobic respiration by Geobacter sulfurreducens

• Electron acceptor: Fe³⁺

• Reduced products: Fe²⁺

Shewanella oneidensis

Uses multiple electron acceptors

Steps to transfer electrons to TEA

1. An electron donor supplies electrons to the ETC in the membrane.

2. The electrons pass to a reductase, an enzyme that transfers electrons to the final acceptor.

3. The acceptor gains electrons and is reduced. The respiratory process helps create a proton gradient that powers ATP production.

Molybdopterin reductases

• Molybdopterin cofactor

• Reduce e^- acceptors such as nitrate, DMSO

Flavin reductases

• FAD or FMN cofactor

• Reduce e^- acceptors such as fumarate

Extracellular Electron Transfer

Process where microbes exchange electrons with solid materials outside the cell - such as metal oxides or artificial electrodes - to drive respiration or energy conservation

Extracellular Electron Transfer Methods

• Direct Extracellular Electron Transfer

  • Redox protein: a protein on the cell surface transfers electrons to an acceptor touching the cell

  • Nanowire: a conductive structure carries electrons from the cell to an acceptor farther away

    • “e^- tube”

    • lots of heme units

• Indirect Extracellular Electron Transfer

  • Mediated electron transfer: mobile soluble carrier picks up electrons from the cell, carries them to the acceptor, and returns for more

Fermentation

• Some bacteria cannot make an ETC, so they rely on fermentation to produce ATP

• Microbes perform any sort of respiration first (if possible) until all alternative electron acceptors are depleted

• Oxygen is not needed

• ATP formed by substrate level phosphorylation

• Max ATP yield = 4

Big problem of fermentation: how do you re-oxidize NADH without using the ETC?

• Solution: dump electrons onto pyruvate produced by initial oxidation (pyruvate = electron sink)

• Same input molecule serves as both the electron donor and acceptor

• Die of starvation if NADH is not re-oxidized

Fermentation pathways

Named based on end product

Main fermentation pathways

1. Lactic acid (enteric bacteria)

2. Ethanol (yeast)

3. Acetate (enteric bacteria)

4. Ethanol (enteric bacteria)

Alcoholic fermentation

Pyruvate decarboxylated to acetalaldehyde —> reduced to ethanol by alcohol dehydrogenase

Homofermentative: homolactic fermenter

Only makes lactic acid (pyruvate —> lactate via lactate dehydrogenase)

Heterofermentative: heterolactic fermenter

Makes lactic acid and other byproducts e.g. ethanol and CO₂

Mixed acid fermentation

• Excretion of a mixture of acids (acetic, lactic, succinic, formic acids)

• Pyruvate dehydrogenase is repressed

• Pyruvate formate lyase is active

  • Pyruvate —> Acetyl-CoA + Formate

• Gas is produced if the microbe makes formate hydrogen lyase

  • Formate —> CO₂ + H₂

2nd big problem of fermentation: acidification

• Fermenters need to pump protons out to prevent internal acidification and make PMF for transport without an ETC

• Solution: use an ATPase to pump protons into the environment

Biosynthetic issues of fermentation

• The cell still needs TCA intermediates to build molecules, even though it cannot rely on that respiratory pathway to recycle NADH

• The horseshoe TCA cycle is used to make building blocks for the cell

• 2 main branches:

  • Oxidative - Oxaloacetate → citrate → isocitrate → α-ketoglutarate

    • produce precursor for amino acids

  • Reductive - Oxaloacetate → malate → fumarate → succinate

    • consumes reducing power and supplies biosynthetic intermediates

Fermentation vs. Anaerobic Respiration

Energy generation (theoretical max)

• Aerobic - 38

• Anaerobic - 36 or less

• Fermentation - 2

Binomial system

• formal two-part scientific naming system used to identify living organisms

• First capitalized, latinized names

• Genus species

  • Escherichia coli

  • Pseudomonas aeruginosa

• Write full name out first, then shorten

• Carolus Linnaeus - formulated this system

Classification systems (past —> present)

• Phenetic —> Phyolgenetic —> Genotypic

• Phenetic was based on similarities and didn’t take into account evolution

• Phylogenetic was based on evolution

• Genotypic is based on genetic material

Classical characteristics for identification

• Morphology

• Biochemistry

• Metabolism

• Ecology

• Genetic

• Molecular

Carl Woese

• Revealed that despite morphological similarities, archaea represented a totally distinct domain of life

  • His analysis relied instead on 16S rRNA (universal genes) analysis

Use of rRNA

• Helps scientists identify organisms and determine evolutionary relationships

• Bacteria and Archaea: 16S

• Eukarya: 18S

• Conserved regions: similar across many organisms, allowing primers to bind

• Variable regions: differ among organisms, helping scientists distinguish them

• 27F and 1492R are universal primers that amplify the 16S gene in all branches of life

  • You need both on either side for PCR

  • A forward primer (27F) binds near one end

  • A reverse primer (1492R) binds near the other end, on the opposite DNA strand

  • The primers point toward each other, so DNA synthesis copies the region between them. Having both primers lets PCR repeatedly double that region

Archaea

• Methanogens

• Hyperthermophiles

• Extreme halophiles

• Mesophiles

Methanogens (euryarchaeota)

• Use the Wolfe Cycle

• Source of electrons —> CO₂

• What is the acceptor —> H₂

• Environmental conditions —> anaerobic

• CO₂ + 4H₂ —> CH₄ + 2H₂O

• In Methano-bacteriota, halobacteriota, and thermoplasmatatota

Halobacteriota (euryarchaeota)

• Extreme halophilic archaea

• Require at least 1.5M NaCl, optima at 3-4M

Thermoplasmas (euryarchaeota)

• Plasmas lack cell walls

  • No peptidoglycan

• Rely on extensive lipoglycans for membrane stability; some grow at pH 0-1

Thermoproteota

• Pyrodictium spp.

• Can survive the autoclave

Nanoarchaeota

• Nanoarchaeum equitans - parasitic archaeon

  • Lives in hydrothermal vents associated with Ignicoccus hospitalis (thermoproteota)

Asgard Archaea

Closest clade to eukarya (new clade)

Non-proteobacterial Gram (-) bacteria

• Hyperthermophiles

  • Aquificota

  • Thermogota

• Photosynthetic bacteria

  • Chloroflexota

  • Chlorobiota

  • Cyanobacteria

• Bizarre cell walls

  • Deinococcota

• Unique morphologies

  • Spirochaetota

Cyanobacteria

• Come in all shapes and sizes

• Also known as blue-green algae

• Predominantly photosynthetic

Deinococcus species

• Weird envelope

• Stain Gram + but have OM

• No teichoic acid

• Altered PL content

• Highly resistant to radiation b/c of really good DNA repair mechanisms

Planctomycetes

• Highly organized membrane systems

  • Some have membrane-bound organelles

• Anammox (anaerobic ammonium oxidation) bacteria

  • Paryphoplasm: outermost peripheral compartment

  • Riboplasm: central compartment containing ribosomes + nucleoid

  • Anammoxosome: largest internal compartment dedicated to energy metabolism

Anammoxosome

Primary site for the anammox process, where ammonium (NH₄⁺) and nitrite (NO₂^-) are converted directly into diatomic nitrogen gas (N₂) and H₂O

Spirochetes

• Treponema

• Borrelia

• Spirochaeta

• They are shaped the way they are because there are flagella all over the outside

• “Corkscrew itself through environment”

Important member of gut biome

Bacteroides

Proteobacteria

• One of the largest and most diverse phyla of Gram - bacteria

  • Vibrionaceace

  • Nitrifying bacteria

  • Photoheterotrophs

  • Enterobacteriaceae

  • Pseudomonadaceae

  • Rhizobiaceae

  • Caulobacteracaeae

Gammaproteobacteria

• Largest bacterial class

• Enterobacteriaceae

  • E. coli

  • Salmonella enteriditis

• Pseudomonadaceae

  • Pseudomonas aeruginosa

Alphaproteobacteria

• Rhizobiaceae

• Caulobacteracaeae

Caulobacter (alphaproteobacteria)

• Asymmetric cell division

• Developmental cycle leads to different cell morphologies

Nitrobacter (alphaproteobacteria)

• Nitrifying bacteria

  • Oxidize nitrite to nitrate

Agrobacterium (alphaproteobacteria)

Negative association with plants (invade plants and cause tumors)

Rhizobium (alphaproteobacteria)

Positive association with plants (invade leguminous plants to produce nitrogen-fixing root nodules)

Pelagibacterales (alphaproteobacteria)

• SAR11 clade

• Comprise 25-50% of all prokaryotic cells in the world’s oceans

• Main driver of the marine carbon cycle

• Never cultured in lab

Gram + Bacteria

• Firmicutes

  • Low GC

• Actinobacteria

  • High GC

Firmicutes

• 2 very important classes:

  • Clostridia

    • Clostridium spp.

  • Bacilli

    • Bacillus spp.

    • Staphylococcus spp.

    • Lactobacillus spp.

    • Streptococcus spp.

Actinobacteria

• Present in gut microbiome

  • Bifidobacterium

  • Micrococcus

  • Propionibacterium

• Pathogen

  • Mycobacterium

    • Mycobacterium tuberculosis = leading cause of death from a single infectious agent

• Antibiotics

  • Streptomyces

    • Source of up to 80% of naturally occurring, clinically useful antiobiotics

Uncultivated clades

• Many of them!

• Know about them because of sequencing

Biogeochemical cycling

Sum of the microbial, physical, and chemical processes that drive the flow of elements

Abiotic processes

• Nonliving (erosion, sedimentary rock)

  • e.g. phosphorus cycle

Biotic processes

Nutrient cycling

What determines microbial activity?

• Redox potential

  • What products can you reduce and consume?

• Determines available terminal electron acceptors for anaerobic respiration

• Determines what reduced molecules are present for electron donors

Winogradsky column

• Different microbes in each layer

  • Efficiency (energy yield) is highest at the top and decreases as you go down

  • Top = oxic (aerobic respiration)

  • Bottom = anoxic (anaerobic respiration)

• Sergei Winogradsky discovered multiple types of lithotrophy involved in nutrient cycling

Elements important in Biogeochemical Cycling and their Redox States

• Reduced Form: donates electrons

  • Methane: CH₄

  • Ammonium: NH₄⁺; organic N

  • Hydrogen sulfide: H₂S; SH groups in organic matter

  • Ferrous iron: Fe²⁺

  • Phosphonates and phosphite: PO^3-

  • Manganese ion: Mn²⁺; Manganous oxide: MnO

• Oxidized Form: accepts electrons

  • Carbon dioxide: CO₂

  • Nitrate: NO₃^-

  • Sulfate: SO₄^2-

  • Ferric ion: Fe³⁺

  • Polyphosphate, phosphate: PO₄^-

  • Manganese dioxide: MnO₂

Carbon cycle

• Carbon is continuously transformed from one form to another

  • First plants and microbes fix CO₂

  • CO₂ is reduced to CH₄

• Methanogenesis

  • Wolfe Cycle

  • Archaea only, in extreme environment & guts

  • CO₂ +4H₂ —> CH₄ + 2H₂O

  • Also possible from acetate, methanol, and methylated contaminants

• Methanotrophy

  • O₂ required

  • Methanotrophic bacteria + archaea

    • Specific to methane

  • Methylotrophic bacteria + archaea

    • Using single C molecules as electron donor

    • Use methane as source of electrons and O₂ as TEA

  • Anoxic alternatives:

    • One microbe: anaerobic methane oxidation coupled to dentrification- uses CH₄ as e^- donor to reduce nitrate

    • Two microbes: methanotrophic archaea + SO₄^- reducing bacteria do reverse methanogenesis

Degradation of organic matter

• Influenced by several factors:

  • Oxidation-reduction potential

  • Availability of competing nutrients

  • Abiotic conditions

    • pH, temperature, O₂, and osmic conditions

  • Microbial community present

Lignin (organic polymer found in wood and plants)

• Microorganisms secrete hydrolytic enzymes that degrade lignin

  • Only contain carbon, hydrogen, oxygen

• Microbes must acquire remaining nutrients

  • Often difficult because of concentration of nitrogen, phosphorus, and iron may be very low

• The nutrient that is insufficient for maximal growth is said to be limiting

  • Liebig’s law of the minimum: doesn’t matter if you have everything else you need b/c something else may be limiting

Nitrogen cycle

• Nitrogen exists in dynamic redox states from -3 to +5

  • Serve as electron donors or acceptors

• Nitrogen fixation: reduction of inorganic N₂ to organic form

  • Redox neutral —> reduced form

  • Performed by some bacteria and archaea

  • Can be carried out under both oxic and anoxic conditions

  • Product is ammonia NH₃

    • Immediately incorporated into organic matter as amine

• Nitrification: ammonium donates electrons (oxidized to nitrite/nitrate)

  • NH₄⁺ —> NO₂^-

  • NO₂^- —> NO₃^-

• Fate of NO₃^-

  • Assimilatory nitrate reduction: NO₃^- is reduced and incorporated into microbial and plant cell biomass

  • Dissimilatory nitrate reduction: when nitrate is fully reduced to N₂, nitrogen is removed from ecosystem and returned to atmosphere

    • Denitrification

• Anammox reaction

  • Anaerobic

  • NH₄⁺ = electron donor

  • NO₂^- = TEA

  • Shortcut to produce N₂ without having to cycle through nitrate

Well characterized genes associated with nitrogen fixation

nifHDK

Well characterized genes associated with denitrification

narG, nas

Well characterized genes associated with nitrite reduction

nirK