The Origin and Evolution of Heterotrophy

Lecture 7: The Origin and Evolution of Heterotrophy

Introduction

  • The concept of a universal tree provides essential organization to microbial systematics. However, it lacks in mapping physiological evolution due to horizontal gene transfer (HGT).

  • Geochemical isotope evidence contains indications of physiological processes rather than phylogeny, showing that lateral gene transfer decouples physiology from phylogeny in prokaryotes.

  • A comprehensive understanding of microbial evolution necessitates integration of physiology, phylogeny, and geological records.

  • Non-fermentative substrate-level phosphorylations, specifically the WL pathway observable in certain acetogens and methanogens, appear significant for establishing chemical foundations for life's tree.

Fermentation as a Metabolic Process

  • Definition: Fermentation (substrate-level phosphorylation) is an anaerobic metabolic process that utilizes the redox potential of reactants to generate ATP without an electron transport chain, producing organic end-products.

  • Sugars are easily fermented through glycolytic reactions, but there's considerable diversity in fermentative pathways. Various organic substrates besides sugars can be utilized, including amino acids, purines, and pyrimidines.

    • Many organisms fermenting these substrates commonly produce acetate as a by-product.

  • Energy conservation in fermentation allows cells to achieve a final redox balance, such as the balance between NAD+ and NADH. The inclusion of molecular hydrogen (H₂) may enhance redox balance in fermentative processes involving organic compounds.

Mechanism of Fermentation

  • The essential components of fermentation include:

    • Oxidation Process: An organic compound is oxidized, returning electrons to an oxidized organic product due to the absence of an external electron acceptor.

    • Product Excretion: The fermentation product formed is excreted, with ATP produced via substrate-level phosphorylation.

  • Most of the fermentable substrate contributes to energy generation, while a comparatively small fraction is allocated to biosynthesis.

Hydrothermal Vents and Fermentation Dynamics

  • Hydrothermal vents have limited life spans, and when a vent (e.g., Lost City) ceases to flow, primary production halts due to the absence of H₂.

  • Without H₂, typical amino acid fermentations, which produce H₂ as a waste product, can become exergonic.

  • Cells primarily composed of proteins (50-60% by dry weight) create opportunities for fermenters to utilize Leeaches from dead or dying cells.

  • Minimal biochemical adaptations are necessary for chemolithoautotrophs to evolve into amino acid fermenters, with existing amino acid synthesis pathways remaining reversible and functional in the H₂ deficit.

Peptidases and Amino Acid Fermentation

  • Peptidases involved in housekeeping within autotrophs can be repurposed into a general secretory pathway, digesting environmental proteins.

  • The resultant amino acids or small peptides may then be imported, providing carbon, nitrogen, and energy sources.

  • The energy conservation process in typical amino acid fermentation includes:

    • Breakdown of the amino acid into corresponding α-keto acids (also denoted as 2-oxo acids) and ammonia through deamination.

    • Subsequently, the α-keto acids undergo oxidative decarboxylation, generating acetyl-CoA as a precursor for acetate synthesis and ATP production.

    • Ammonia serves as a nitrogen source for cellular growth.

Bacterial and Archaeal Fermentation of Amino Acids (Heterotrophy)

  • Amino acids, phosphate (Pi), ammonia (NH₃), acetyl-CoA, and products like acetate and α-keto acids feature in bacterial and archaeal fermentation processes.

  • Anaerobic autotrophs are considered the progenitors of the first heterotrophs, offering substrates for their growth, with fermentations yielding approximately 1 ATP per amino acid.

  • Example: Clostridium tetanomorphum (a strict anaerobe) generates 1 ATP by fermenting glutamate into acetate, butyrate, CO₂, and H₂.

Stickland Fermentation

  • Stickland fermentation refers to reactions that couple the oxidation and reduction of amino acids resulting in organic acids:

    • Example: Clostridium sporogenes oxidizes alanine while reducing glycine, leading to the production of acetyl-phosphate and ammonia.

  • The transition steps include:

    • Alanine → Pyruvate → Acetyl-P → ATP + NH₃ + CO₂

    • Glycine reacts → Acetyl-P → ATP + NH₃

Special Cases in Stickland Reactions

  • Certain Stickland reactions allow the same amino acid to serve as both donor and acceptor.

  • Example: Clostridium propionicum ferments three moles of alanine to yield three moles of ammonia, one mole of acetate, and two moles of propionate, resulting in the production of 2 ATP.

Amyloid Fermentation in Archaea

  • Among archaea, amino acid fermentation is extensive within the Thermococcales class, where thermophiles thrive in high-temperature environments (80-100 °C), predominantly around hydrothermal ecosystems.

  • The major contrast between bacterial and archaeal amino acid fermentation lies in the enzymatic pathways for energy conservation.

Fermentation of Nucleic Acids

  • Approximately 25% of prokaryotic cell mass consists of nucleic acids; hence fermentation of nucleotides can yield energy or contribute to new nucleic acid synthesis.

  • Example: Clostridium acidiurici can produce 1.25 moles of ATP from 1 mole of uric acid, a metabolite of purine degradation.

Hydrolytic Deamination and Subsequent Fermentation

  • Cytosine transforms into uracil via hydrolytic deamination, a spontaneous process in aqueous environments, followed by the reduction to dihydrouracil.

  • This can further decompose to β-alanine, which Clostridium propionicum can convert to acetyl-CoA and subsequently to acetate, allowing for ATP generation.

Fermentation of Ribose

  • Prokaryotes usually contain around 20% RNA with ribose making up 40% of the total weight. Ribose serves as an excellent energy-dense substrate that can be fermented, facilitated by the pentose phosphate pathway (PPP) in bacteria; archaea utilize the ribulose monophosphate (RuMP) pathway.

  • The PPP is generally absent in archaea, limiting pentose fermentation mostly to facultative anaerobes like Haloarchaea.

Phosphorolysis of Nucleotides

  • The enzymatic process of phosphorolysis cleaves nucleotides, yielding ribose bisphosphate, which is converted to ribulose bisphosphate. By incorporating CO₂ into ribulose bisphosphate, type III RubisCO forms two 3-phosphoglycerates, a pivotal intermediate in carbon metabolism.

Fermentability of Lipids

  • Although considered generally nonfermentable, lipids are believed to have accumulated in primordial environments. These may have been repurposed by the first heterotrophs for membrane construction until oxidative respiration was developed.

The Role of Energy-converting Hydrogenase (Ech) Complex

  • The Ech complex functions as a proton pump in microbes, linking the oxidation of reduced ferredoxin to proton reduction for hydrogen gas formation.

    • Process Details:

    • The reduction of protons (H⁺) generates H₂ and simultaneously, the complex pumps protons outside the cell, creating a transmembrane proton gradient.

    • This gradient is then harnessed for ATP production by ATP synthase.

Limitations of Amino Acid Fermentation

  • The energy yield from amino acid fermentations restricts the biomass production by fermenters, as one ATP is gained per amino acid while biosynthesis of proteins necessitates 4 ATP per peptide bond and an additional 1 ATP per amino acid imported.

  • Consequently, five amino acids sourced from decaying cells are necessary to form a single new peptide bond in a growing cell.

  • Fermentation of ancient cellular remains results in an accumulation of nonfermentable substrates, like lipids and fatty acids, prompting the onset of anaerobic respiration to resume metabolic activities using external electron acceptors.

Introduction to Heterotrophy

  • Understanding the "tree of life" helps organize tiny living things (microbes). However, it doesn't clearly show how their ways of getting food (physiology) changed over time, mainly because microbes often share genes (horizontal gene transfer, HGT).

  • Clues from geological records, like isotopes, show how these processes happened, often separately from how microbes are related genetically.

  • To truly understand how microbes evolved, we need to look at their food-getting methods, their genetic relationships, and geological evidence together.

  • Simple chemical reactions that don't involve oxygen, like the WL pathway found in some acetogens and methanogens, were likely very important for the earliest forms of life.

Fermentation: A Way to Get Energy Without Oxygen

  • What it is: Fermentation is a process cells use to make energy (ATP) without needing oxygen or an electron transport chain. It uses the chemical energy in food molecules and produces organic waste products.

  • Sugars are common foods for fermentation, but many other things like amino acids, purines, and pyrimidines can also be fermented.

  • Many microbes that ferment these non-sugar foods often produce acetate as a byproduct.

  • Fermentation helps cells balance their internal chemistry, like the balance between NAD+ and NADH. Adding hydrogen gas (H_2) can also help this balance when organic compounds are fermented.

How Fermentation Works

  • Key steps:

    • Oxidation: A food molecule is broken down (oxidized), and the electrons are returned to another part of the same food molecule because there's no oxygen or external electron acceptor.

    • Waste Product: The waste product created during fermentation is released, and energy (ATP) is made directly from the food molecule (substrate-level phosphorylation).

  • Most of the food molecule goes towards making energy, with a smaller part used for building cell components.

Hydrothermal Vents and Early Fermentation

  • Places like hydrothermal vents (deep-sea cracks that release hot, chemical-rich water) don't last forever. When a vent stops flowing, the main source of energy (like H_2) disappears.

  • Without H2, the breakdown of amino acids (which normally produce H2 as waste) becomes more favorable for energy production.

  • When cells die at these vents, their proteins (which make up a large part of cells) can be used by other microbes as food.

  • Microbes that originally made their own food (chemolithoautotrophs) could easily switch to fermenting amino acids because they already had pathways for making amino acids, which could be reversed to break them down when H_2 was scarce.

Peptidases and Using Amino Acids for Energy

  • Cells have enzymes called peptidases that normally help manage their internal proteins. These can be adapted to break down proteins from the environment (dead cells).

  • The resulting amino acids or small protein pieces can then be taken in by the cell to get carbon, nitrogen, and energy.

  • How amino acids are fermented for energy:

    • Amino acids are broken down into simpler acids (α-keto acids) and ammonia through a process called deamination.

    • These α-keto acids are then further broken down to produce acetyl-CoA, which is used to make acetate and ATP.

    • The ammonia can be used by the cell as a source of nitrogen for growth.

Amino Acid Fermentation in Bacteria and Archaea

  • Bacteria and archaea use amino acids, phosphate (Pi), ammonia (NH_3), and acetyl-CoA in their fermentation processes, creating products like acetate and α-keto acids.

  • It's thought that the first microbes to eat other things (heterotrophs) evolved from those that made their own food (anaerobic autotrophs), using their dead remains as a food source. These fermentations typically yield about 1 ATP per amino acid.

  • Example: Clostridium tetanomorphum, an organism that cannot live with oxygen, makes 1 ATP by fermenting glutamate into acetate, butyrate, carbon dioxide (CO2), and hydrogen (H2).

Stickland Fermentation: Teamwork in Fermentation

  • Stickland fermentation involves coupling the breakdown (oxidation) of one amino acid with the gain of electrons (reduction) by another amino acid, leading to organic acids.

  • Example: Clostridium sporogenes breaks down alanine while also modifying glycine. This leads to the production of acetyl-phosphate, ammonia, and ATP.

  • The process looks like this:

    • Alanine → Pyruvate → Acetyl-P → ATP + NH3 + CO2

    • Glycine reacts → Acetyl-P → ATP + NH_3

Special Stickland Reactions

  • In some cases, the same amino acid can act as both the giver and receiver of electrons.

  • Example: Clostridium propionicum ferments three molecules of alanine to produce three molecules of ammonia, one molecule of acetate, and two molecules of propionate, yielding 2 ATP.

Amino Acid Fermentation in Archaea

  • Many archaea, particularly those in the Thermococcales class, ferment amino acids. These organisms are thermophiles, meaning they love high temperatures (80-100 \text{ °C}) and are often found around hydrothermal vents.

  • The main difference between how bacteria and archaea ferment amino acids is in the specific enzymes and chemical steps they use to make energy.

Fermentation of Nucleic Acids

  • Nucleic acids (like DNA and RNA) make up about 25% of a prokaryotic cell's weight. Fermenting their building blocks (nucleotides) can provide energy or materials for new nucleic acids.

  • Example: Clostridium acidiurici can get 1.25 moles of ATP from 1 mole of uric acid, which is a breakdown product of purines.

Breaking Down Nucleic Acid Components

  • Cytosine naturally changes into uracil in water. Uracil can then be reduced to dihydrouracil.

  • This can further break down into β-alanine, which Clostridium propionicum can convert into acetyl-CoA and then acetate, generating ATP.

Fermentation of Ribose

  • RNA, which contains ribose sugar, makes up about 20% of prokaryotic cells. Ribose is an excellent energy source for fermentation.

  • Bacteria use the pentose phosphate pathway (PPP) for this, while archaea use the ribulose monophosphate (RuMP) pathway.

  • The PPP is generally not found in archaea, so most archaeal pentose fermentation is limited to facultative anaerobes (those that can grow with or without oxygen) like Haloarchaea.

Using Ribose for Energy and Carbon

  • Enzymes can break down nucleotides, producing ribose bisphosphate, which then becomes ribulose bisphosphate.

  • By adding CO_2 to ribulose bisphosphate, a special enzyme called type III RubisCO creates two molecules of 3-phosphoglycerate, which is a key molecule in carbon metabolism.

Fermenting Lipids

  • Lipids (fats) are generally hard to ferment. However, it's thought that in early environments, lipids might have accumulated and been used by the first heterotrophs to build cell membranes, before cells developed ways to use oxygen for energy.

The Energy-converting Hydrogenase (Ech) Complex

  • The Ech complex in microbes acts like a pump, taking electrons from a molecule called reduced ferredoxin and using them to create hydrogen gas (H_2) from protons.

  • How it works:

    • It converts protons (H^+) into H_2 gas.

    • At the same time, it pumps protons out of the cell, building up a difference in proton concentration across the cell membrane.

    • This proton difference (gradient) is then used by another enzyme (ATP synthase) to make ATP.

Limits of Amino Acid Fermentation

  • The amount of energy (ATP) obtained from amino acid fermentation is relatively low (about one ATP per amino acid).

  • Building new proteins, however, requires a lot of energy: 4 ATP per peptide bond (the link between amino acids) and an additional 1 ATP per amino acid imported into the cell.

  • This means a cell needs to break down about five amino acids from dead cells to create just one new peptide bond in its own growing proteins.

  • Over time, fermentation of dead cells leaves behind molecules that are hard to ferment, like lipids and fatty acids. This eventually forces microbes to switch to anaerobic respiration, a process that uses external electron acceptors instead of relying solely on internal food molecules, to continue getting energy and growing.