City College of San Francisco Microbiology 12

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MICROBIAL METABOLISM

Prokaryotes can be organized into groups based upon their nutritional and metabolic needs which are extremely diverse. Traditionally, these groupings have been based on two main criteria:

the nature of the energy source

the nature of the carbon source used for building organic, biological macromolecules

METABOLISM is defined as the sum of all chemical reactions occurring within a living organism. CATABOLIC reactions are energy-releasing (exergonic) reactions which break down more complex molecules, usually by hydrolysis, into simpler components. The chemical processes of digestion typically occur by this route. ANABOLIC reactions are energy-requiring (endergonic) and build more complex molecules, usually by condensation, from subunit components. The energy for anabolic reactions is provided by catabolic reactions; they are ALWAYS LINKED. Energy is ultimately stored in the form of the energy -rich molecule, ATP.

Our focus is on the metabolic pathways used by bacteria to generate their energy requirements. To illustrate this point we will follow the fate of the energy in glucose as it is catabolized by:

• Glycolysis (a preparatory, anaerobic process)
• The Krebs cycle (generation of energy-carriers)
• The electron transport system (to produce ATP either by aerobic or anaerobic conditions)
• Fermentation pathways (following glycolysis)

Before we follow these pathways in more detail we need to review the major steps involved in energy production:

Bacteria use three main mechanisms of PHOSPHORYLATION to produce ATP

Energy released from certain metabolic reactions can be trapped to form ATP from ADP and a phosphate group by a process called phosphorylation. This chemical reaction is catalyzed by an enzyme called ATP synthetase.

Substrate level phosphorylation: occurs when ATP is formed directly by the addition of a phosphate to ADP. Occurs both in glycolysis and the Krebs cycle.

Oxidative phosphorylation: energy in the form of electrons is released stepwise from oxidized organic compounds (e.g. glucose) to electron carriers (usually NAD+ or FADH). Electron carriers enter a membrane-associated electron transport system (ETS). The ETS and ATP synthetase occur on the inner mitochondrial membrane of eukaryotes and on the plasma membrane of prokaryotes. Electrons are ultimately donated to final electron acceptors to form ATP. The electron acceptors include:

• molecular oxygen (aerobic respiration)
• an inorganic molecule other than oxygen (anaerobic respiration)

Photophosphorylation: occurs only in photosynthetic organisms which trap light energy by photosynthetic pigments and convert it to the chemical energy of ATP. This process involves an electron transport system. Photosynthetic bacteria include cyanobacteria and the green and purple sulfur bacteria . Cyanobacteria lack chloroplasts but possess photosynthetic membranes called thylakoids. The thylakoids possess the photosynthetic pigments chlorophyll a, carotenoids and phycobiliproteins. The green and purple sulfur bacteria possess bacteriochlorophyll.

What is meant by OXIDATION and REDUCTION?!

In my experience, these two concepts often give students a great deal of trouble. Oxidation and reduction reactions (also known as REDOX reactions) are always coupled in biological systems.

Put quite simply, oxidation reactions release energy. Compounds that contain the greatest amount of stored chemical energy are hydrocarbons such as fats and lipids. In biological systems, oxidation typically involves:

• the loss of hydrogen atoms (equivalent to protons) from a substrate
• loss of hydrogen atoms are known as dehydrogenation reactions
• electrons are typically lost together with hydrogen atoms
• the addition of oxygen is also termed oxidation
• an "oxidized" molecule has given up energy. For example, the energy carrier molecule NAD+, is an energy-deficient form of an electron carrier as it has given up a hydrogen and two electrons.

Reduction reactions harness chemical energy. Reduction involves:

• the gain of electrons and hydrogen atoms by a substrate. This is what confuses people; how can a gain of something be termed "reduction?" The answer is that:
• the loss of oxygen is also termed reduction
• a "reduced" molecule is energy rich. For example, NADH picks up 2 energetic electrons and a hydrogen atom.

These reactions are always coupled as the electrons lost from an oxidized molecule have to be transferred to another. A source of electrons, or electron donor, is referred to as a reducing agent, while the electron acceptor is the oxidizing agent as it oxidizes some other molecule and becomes reduced in so doing.

GLYCOLYSIS

Glycolysis literally means "sugar splitting." It is the most common pathway for the breakdown or oxidation of glucose and is typically the first stage in carbohydrate catabolism.

• Involves 10 distinct chemical reactions, each catalyzed by different enzyme. Glucose is split into 2 molecules of pyruvic acid and is accompanied by ATP formation through substrate level phosphorylations.
• Glycolysis (Embden-Meyerhof Pathway) is used by eukaryotic cells, many anaerobic and facultatively anaerobic bacteria. The Archaea do not use this pathway.

4 key events occur during glycolysis:

• During a preparatory stage, glucose becomes energized by the addition of two phosphate groups. This step also alters the conformation of glucose and renders it incapable of leaving the cell. Two molecules of ATP are used to energize glucose. The resulting fructose 1,6 diphosphate is subsequently split into two, three-carbon compounds.
• Substrate level phosphorylation yields 2 molecules of ATP directly from ADP. As two phosphorylated 3-carbon molecules are formed from each molecule of glucose 4 molecules of ATP are actually formed at this point. There is a net yield of 2 ATP per glucose entering glycolysis.
• For each three carbon compound a molecule of NAD+ is reduced to a molecule of NADH. The energy rich molecules of NADH can enter the electron transport system so that more ATP can be generated by oxidative phosphorylation.
• Two molecules of pyruvate result which. Pyruvate may be fermented or enter the Krebs cycle depending on the bacterium.

Many aerobic bacteria, such as Pseudomonas spp, and the Archaea use an alternative glycolytic pathway called the Entner-Doudoroff pathway. This pathway produces pyruvate directly from glucose and lacks some of the ATP-generating steps found in the Embden-Meyerhof pathway. Only 1 ATP is gained from each molecule of glucose.

Regardless of the pathway used, glycolysis as an energy-yielding pathway is acceptable for simple organisms such as yeast. However, only 2% of energy is in the form of ATP; the rest is distributed between pyruvate and heat.

THE KREBS CYCLE

The purpose of the Krebs cycle is to harness useful energy in the form of electrons, carried by the reduced coenzymes, NADH and FADH₂.

Key events of the Krebs cycle:

• Each molecule of pyruvate from glycolysis is decarboxylated (loss of CO₂) to become a 2 carbon compound (acetyl group) which attaches to coenzyme A (acetyl CoA).
• The 2 carbon acetyl groups enter the cycle and pass through a series of reactions whereby chemical energy is released stepwise.
• The carbons that enter the Krebs cycle are oxidized to CO₂ and are eliminated as waste
• For each turn of the Krebs cycle four pairs of electrons are transferred to coenzymes; 3 pairs reduce NAD+ to form three molecules of NADH and 1 pair of electrons reduce FADH to FADH2. Energy is extracted from these coenzymes as they enter the electron transport system.
• For each turn of the Krebs cycle (two turns), substrate level phosphorylation generates the equivalent of one ATP.

ELECTRON TRANSPORT CHAIN

• The carriers of the electron transport system occur on the plasma membrane of bacteria.
• Energy carrying molecules of NADH and FADH2 donate their electrons to a series of coenzymes. The electrons are ultimately donated to a terminal electron acceptor, which in the case of aerobic respiration, is oxygen. The electrons are then are reunited with protons to form water.
• Each carrier is reduced as it picks up electrons and oxidized as it passes them on (gives up energy)
• Most ATP generated by aerobic respiration is by oxidative phosphorylation:
• occurs at 3 sites at ETS so ADP is phosphorylated to ATP
• FAD enters lower down the ETS and only 2 sites are utilized for this coenzyme
•  
• A TOTAL OF 38 ATP is PRODUCED BY AEROBIC RESPIRATION IN PROKARYOTES

CHEMIOSMOSIS

How energy captured to form ATP.

This theory was formulated by the British biochemist, Peter Mitchell, who won the Nobel prize 1978. This theory proposes the mechanism whereby the electron transport system siphons off electrons from the Krebs cycle and uses the energy to produce ATP by oxidative phosphorylation.

A molecule of NADH entering the ETS donates a pair of electrons to the first coenzyme in the pathway. For each molecule of NADH, three pairs of hydrogen ions (protons) are pumped across the inner mitochondrial membrane into the intermembrane space (of eukaryotes). This generates a concentration and electrical gradient between the membrane space and mitochondrial matrix. Protons diffuse back down this concentration gradient through channels in the inner membrane into the mitochondrial matrix. ATP synthetase, associated with the channels, phosphorylates ADP to ATP. Each pair of hydrogens drives formation of a molecule of ATP.

A rule of thumb is that for each molecule of NADH entering this system, three molecules of ATP are produced. FADH₂ enters the ETS further down the pathway at coenzyme Q and donates two pairs of electrons. Consequently, each molecule of FADH₂ only gives rise to 2 ATP molecules.

FERMENTATION

Fermentation can have a variety of meanings, ranging from informal to more scientific definitions. The various meanings of fermentation are nicely summarized by Tortora et al ( Microbiology, 6th Ed, 1998):

Any spoilage of food by microbes. For example, the spoilage of wine to vinegar. This is a very general usage of fermentation

Any process that produces alcoholic beverages or acidic dairy products (again general use)

Any large scale microbial process occurring with or without air (industrial use)

Any energy-releasing process that occurs only under anaerobic conditions (more scientific)

Any metabolic process that releases energy from a sugar or other organic molecule, does not need oxygen or an electron transport system, and uses an organic molecule as the final electron acceptor. It is this last definition that we will use.

Some other key points that we need to keep in mind are:

• A complete fermentation pathway begins with a substrate, includes glycolysis and results in various end-products. The different fermentation pathways typically are named for the end products that are formed.
• As far as an energy is concerned, fermentation does not generate ATP directly but recycles a limited amount of NAD+ back into glycolysis to keep glycolysis going. Recall that each pass through glycolysis generates 2 ATP molecules by substrate level phosphorylation.
• All fermentation pathways are anaerobic.
• Cells that are capable of both respiration and fermentation will typically use respiration when possible. Respiration yields more energy from a lot less substrate.