Metabolic diversity in Bacteria

Basically three major energy, electron and carbon deriving systems are present in the bacterial world.

Chemo-organotrophic - heterotrophs: Derive all their requirements from organic compounds.
Photo-lithotrophic-autotrophs: Derive from sunlight and CO2 fixation.
Chemo-lithotrophic-autotrophs: Derive from inorganic chemicals and CO2 fixation.

In this chapter, we will see, how the metabolic processes take place in these bacteria.

Chemo-oranotrophic-heterotrophs

Those organisms depend on organic compounds as source of energy, electron and carbon. Example Escherichia coli, Salmonella, Rhizobium, Azospirillum, Lactobacillus like bacteria and all fungi (including yeast). These organisms perform two types of reactions for their energy production from these organic compounds viz., fermentation and respiration. Fermentation is the form of anaerobic catabolism in which an organic compound is both an electron donor and an electron acceptor, and ATP is produced by substrate-level phosphorylation. Respiration is the catabolism in which a compound is oxidized with O2 (or any other substitute) as the terminal electron acceptor and ATP is produced by oxidative phosphorylation. In both series of reactions, ATP synthesis is coupled to energy released in oxidation–reduction reactions.

Fermentation and respiration are the alternative metabolic choices available to some microorganisms. In organisms that can both ferment and respire, such as yeast, fermentation is necessary when conditions are anoxic and terminal electron acceptors are absent. When O2 is available, respiration can take place. More ATP is produced in respiration than in fermentation and thus respiration is the preferred choice. But many microbial habitats lack O2 or other electron acceptors that can substitute for O2 in respiration, and in such habitats, fermentation is the only option for energy conservation by chemoorganotrophs.

Glucose: Respiration and Fermentations

We will consider glucose, the high energy compound which can be utilized by most of the bacteria present in the environment. Upon transport into the cytoplasm, most of the bacteria oxidize the glucose to pyruvate through a series of metabolic reactions or pathway known as glycolysis. The pathway is also referred as Embden–Meyerhof–Parnas pathway. Glycolysis has three stages.

Stage I: No change in redox reactions. Preparation for oxidation

During this stage, glucose is phosphorylated by ATP, yielding glucose 6-phosphate; then isomerized to fructose 6-phosphate. A second phosphorylation leads to the production of fructose 1,6-bisphosphate. The enzyme aldolase then splits fructose 1,6- bisphosphate into two 3-carbon molecules, glyceraldehyde 3-phosphate and its isomer, dihydroxyacetone phosphate, which can be converted into glyceraldehyde 3-phosphate. To this point, all of the reactions, including the consumption of ATP, have proceeded without redox reactions (means no change in their oxido-reductive potentials).

Stage II: Oxidation and ATP and NADH synthesis

In this stage, the glyceraldehyde 3 phosphate is oxidized into 1,3 bisphosphoglycerate. The electrons released were used for reduction of NAD to NADH (enzyme responsible is glyceraldehyde-3-phosphate dehydrogenase). During this reaction, simultaneously, inorganic phosphate also added (phosphorylated) with glyceraldehyde 3 phosphate. This phosphorylation is the step for energy conservation. 1,3 bisphosphoglycerate is converted to 3-phosphoglycerate which yielded one ATP. Further, 3-phosphoglycerate is converted to phosphoenolpyruvate (PEP). The further oxidation of PEP release one ATP and formation of pyruvate.

At the end of glycolysis, two ATP were consumed in stage 1 and four ATPs were produced in stage 2 (which occurs twice for one glucose molecule) along with 2 NADH (reducing power). Thus, net energy yield of glucose molecule is 2 ATP per glucose.

Glycolysis is substrate-level phosphorylation (fermentative)

Please note that the ATP is synthesized through glycolysis without aid of external electron acceptors. Hence it is a substrate-level phosphorylation. Even the organisms with respiratory pathway should have the glycolysis, which is a fermentative pathway.

Stage 3: Fermentative mode of energy conservation by fermentative microorganisms

When 1,3 bisphosphogycerate is oxidised NAD is reduced to NADH (2 NADH per glucose). However, the NADH should be oxidised back to NAD. Why? NAD is the only electron shuttle, not a net (terminal) acceptor of electrons. Thus, the NADH produced in glycolysis must be oxidised back to NAD in order for glycolysis to continue, and this is accomplished when pyruvate is reduced (by NADH) to fermentation products. So several bacteria and fungi prefer this bypass rather than complete oxidation (respiration), even though the ATP yield is relatively very low. For example, in yeast, pyruvate is reduced to ethanol. By contrast, lactic acid bacteria reduce pyruvate to lactate. Many other possibilities for pyruvate reduction are possible depending on the organism, but the net result is the same: NADH is re-oxidized to NAD during the production of fermentation products, allowing reactions of the pathway that depend on NAD to continue.

During glycolysis, glucose is consumed, two ATPs are made and fermentation products are generated. For the organism the crucial product is ATP, which is used in energy-requiring reactions; fermentation products are merely waste products. However, fermentation products are not considered wastes by human. So, fermentation is more than just an energy-yielding process for a cell; it is also a means of making natural products useful to humans.

Pasteur Effect: When glucose is consumed by yeast, it showed variation in the alcohol formation (fermentation). The key factor responsible for the alcohol formation is the supply of oxygen. Pasteur described as “the ferment (yeast) lost its fermentative abilities in proportion to the concentration of this gas”. This phenomenon in which aeration of yeast broth increases the cellular growth not the fermentative product (alcohol) is known as ‘Pasteur effect’.

Oxidative mode of energy conservation by respiratory microorganisms

In contrast to the fermentative pathway, if O2 (or other usable terminal acceptors) are present, pyruvate can be further oxidized to CO2 instead of being reduced to fermentation products and excreted. When pyruvate is oxidized to CO2, a far higher yield of ATP is possible. Oxidation using O2 as the terminal electron acceptor is called aerobic respiration; oxidation using other acceptors under anoxic conditions is called anaerobic respiration.

If an organism further precedes the glycolysis through respiratory mode of energy production, the organism needs the followings:

Tricarboxylic acid (TCA) / Citric acid / Krebs cycle
Membrane-bound electron transport chain
External terminal electron acceptor
Membrane bound ATPase enzyme

During respiration, we should know two important events take place.

1. Pyruvate (3-carbon compound) undergone further oxidation into 3 carbon-di-oxide through pathway of reactions;

2. The electrons released during this process were transported by several carriers and finally to terminal electron acceptor (O2) which is coupled to energy generation by ATPase enzyme.

We will see the processes in detail in this chapter.

TCA cycle

The pathway by which pyruvate is completely oxidized to CO2 is called the citric acid cycle or TCA cycle or Krebs cycle.

Naming: Since citric acid is the primary and first product of the cycle and it is a three carboxylic acid (-COOH) group containing compound, it is called as TCA cycle or citric acid cycle. This cycle was named in recognition of the German chemist Hans Krebs, whose research into the cellular utilization of glucose contributed greatly to the modern understanding of this aspect of metabolism.

Over-all reaction: Pyruvate is first decarboxylated, leading to the production of CO2, NADH, and the energy-rich substance acetyl-CoA. The acetyl group of acetyl-CoA then combines with the four-carbon compound oxalacetate, forming the six-carbon compound citric acid. A series of reactions follow, and two additional CO2 molecules, three more NADH and one FADH are formed. Ultimately, oxalacetate is regenerated to return as an acetyl acceptor, thus completing the cycle.

The oxidation of pyruvate to CO2 requires the concerted activity of the citric acid cycle and the electron transport chain. For each pyruvate molecule oxidized through the citric acid cycle, three CO2 molecules are released. Electrons released during the oxidation of intermediates in the citric acid cycle are transferred to NAD to form NADH, or to FAD to form FADH2. This is where respiration and fermentation differ in a major way. Instead of being used in the reduction of pyruvate as in fermentation, in respiration, electrons from NADH and FADH2 are fuel for the electron transport chain, ultimately resulting in the reduction of an electron acceptor (O2) to H2O. This allows for the complete oxidation of glucose to CO2 along with a much greater yield of energy. Whereas only 2 ATP are produced per glucose fermented in alcoholic or lactic acid fermentations, a total of 38 ATP can be made by aerobically respiring the same glucose molecule to CO2 + H2O.

Electron transport chain

In respiratory organisms, the cytoplasmic membrane is energized by proton motive force created through series of electron carriers. This event is called as ‘electron transport chain’. Let us see how it helps the ATP synthesis through oxidative phosphorylation.

We must first understand how the electron transport system is oriented in the cytoplasmic membrane. Electron transport carriers are oriented in the membrane in such a way that, as electrons are transported, protons are separated from electrons. Two electrons plus two protons enter the electron transport chain from NADH through NADH dehydrogenase to initiate the process. Carriers in the electron transport chain are arranged in the membrane in order of their increasingly positive reduction potential, with the final carrier in the chain donating the electrons plus protons to a terminal electron acceptor such as O2.

Step I: NADH dehydrogenase bound to the inside surface of the cytoplasmic membrane. They have an active site that binds NADH and accepts two electrons plus two protons (2 e- and 2 H+) when NADH is oxidized to NAD+. Both 2 e- and 2 H+ were then transferred to flavoprotein.

Step 2: Flavoproteins are derivative of the vitamin riboflavin. It accepts 2 e- and 2 H+ and oxidized when 2 e- are passed on to the next carrier in the chain, Fe/S protein. Note that flavoproteins accept 2 e- and 2 H+ but donate only electrons. So, four H+ ions accumulated in both the steps will be extruded to the outer surface now.

(Note: NADH dehydrogenase, flavoprotein and Fe/S protein represents complex I of the electron transport chain).

Step 3: Now, 2e- and 2H+ from Fe/S protein were accepted by quinone (denoted as Q) and reduced to QH2. Now, like Fe/S protein, quinone also transfer the 2e- to the next carrier and four H+ (already accepted two and newly generated two) were extruded to the outer space.

Step 4: The second complex (Referred as complex II) which also produce the protons and electrons (as that of complex I) is succinate dehydrogenase complex. It binds with succinate and oxidized to fumarate and 2e- and 2H+, intermediated by FAD (first reduced to FADH2 and then to FAD) will be pooled to quinone (Step 3). If external FADH2 enters to electron transport, it bypasses the Complex-I and moved to Complex-II.

Step 5: The coenzyme Q (reduced Q or QH2) now donates electron to complex III known as Cytochrome bc1 complex. The bc1 complex is present in the electron transport chain of almost all organisms that can respire. It also plays a fundamental role in photosynthetic electron flow of phototrophic organisms. The major function of the cytochrome bc1 complex is to transfer e- from quinones to cytochrome c. Cytochrome c is not present in the membrane and it is in the periplasmic space of the cell. It acts as a shuttle to transfer e- to the high-potential cytochromes a and a3 (Complex IV).

Step 6: Complex IV is the terminal oxidase and reduces O2 (Terminal electron acceptor) to H2O in the final step of the electron transport chain. Complex IV also pumps (2 H+) protons to the outer surface of the membrane, thereby increasing the strength of the proton motive force.

[Note: If NADH2 enters to the electron transport chain, 4 H+ (Complex I to quinone); 4 H+ (Coenzyme Q cycling); 2H+ (Complex IV). So a total of 10 protons will be pumped out and made the proton-rich periplasmic space. In case of FADH2 enters, it will extrude twice only (4H+ and 2H+)].

Proton motive force: The result of electron transport is thus the formation of an electrochemical potential across the membrane. This potential, along with the difference in pH across the membrane, is called the proton motive force (PMF) and causes the membrane to be energized much like a battery. Some of the potential energy in the PMF is then conserved in the formation of ATP.

ATPase enzyme complex: The ATPase is an enzyme complex consists of F1 and F0 sets of subunits present in the membrane. These subunits are motors. The PMF creates torque in this large protein complex that makes ATP. PMF-driven H+ movement through F0 causes rotation of its proteins. This generates a torque that is transmitted to F1 via the coupled rotation causes conformational changes in the subunits that allows them to bind ADP + Pi. ATP is synthesized when the subunits return to their original conformation, releasing the free energy needed to drive the synthesis. ATPase-catalyzed ATP synthesis is called oxidative phosphorylation if the proton motive force originates from respiration reactions and photophosphorylation if it originates from photosynthetic reactions. Three to four H+ was consumed by ATPase per ATP produced.

Photo – lithotrophic – autotrophs

Phototrophy – the use of light energy for the energy requirement – is also wide spread in bacterial system. The most important biological process on Earth is photosynthesis, the conversion of light energy to chemical energy. Organisms that carry out photosynthesis are called phototrophs and most phototrophic organisms are also autotrophs, capable of growing with CO2 as the sole carbon source. Energy from light is used in the reduction of CO2 to organic compounds (photoautotrophy). However, some phototrophs use organic carbon as their carbon source; this lifestyle is called photoheterotrophy. Photosynthesis requires light-sensitive pigments, the chlorophylls, found in plants, algae, and several groups of prokaryotes. Sunlight reaches phototrophic organisms in packets of energy called quanta. Absorption of light energy by chlorophylls begins the process of photosynthetic energy conversion, and the net result is chemical energy, ATP.

Photoautotrophy requires that two distinct sets of reactions operate in parallel: (1) ATP production and (2) CO2 reduction to cell material. For autotrophic growth, energy is supplied

from ATP and electrons for the reduction of CO2 come from NADH (or NADPH). The latter are produced by the reduction of NAD+ (or NADP+) by electrons originating from various electron donors. Some phototrophic bacteria obtain reducing power from electron donors in their environment, such as reduced sulfur sources, for example hydrogen sulfide (H2S), or from hydrogen (H2). By contrast, green plants, algae, and cyanobacteria use electrons from water (H2O) as reducing power. The oxidation of H2O produces molecular oxygen (O2) as a by-product. This process is called photolysis of water. Because O2 is produced, photosynthesis in these organisms is called oxygenic photosynthesis. However, in many phototrophic bacteria H2O is not oxidized and O2 is not produced, and thus the process is called anoxygenic photosynthesis. Oxygen originally produced on Earth by the oxygenic photosynthesis of cyanobacteria.

Light harvesting pigments

A. Chlorophylls: Chlorophyll (oxygenic phototrophs) and bacteriochlorophyll (anoxygenic phototrophs) are the predominant light-harvesting systems present in the photosynthetic organisms. Chlorophylls contain magnesium in the centre. Chlorophyll-a, the predominant chlorophyll of algae, cyanobacteria and plants is green in color because it absorbs red and blue light preferentially and transmits green light. It absorbs 680 nm wavelength light while chlorophyll-b absorbs 660 nm. The bacteriochlorophyll absorbs 800-920 nm wavelength light.

B. Carotenoids: Carotenoids are hydrophobic light-sensitive pigments that are firmly embedded in the photosynthetic membrane. Carotenoids are typically yellow, red, brown, or green in color and absorb light in the blue region of the spectrum. Carotenoids are closely associated with chlorophyll or bacteriochlorophyll in photosynthetic complexes, and energy absorbed by carotenoids can be transferred to the reaction center. Β carotene, lycopene, xanthophyll are some of the carotenoids well known.

C. Phycobiliproteins: Cyanobacteria and the chloroplasts of red algae contain phycobiliproteins, which are the main light-harvesting systems of these phototrophs. Phycoerythrin (red) absorbs 550 nm light and phycocyanin (blue) absorbs 620 nm light.

Bacterial photosynthesis (Anoxygenic photosynthesis)

In the photosynthetic light reactions, electrons travel through a series of electron carriers arranged in a photosynthetic membrane in order of their increasingly more electropositive reduction potential (E0`). This generates a proton motive force that drives ATP synthesis (known as Photophosrylation). Anoxygenic photosynthesis occurs in at least five phyla of Bacteria: the proteobacteria (purple bacteria); green sulfur bacteria; green nonsulfur bacteria; the gram-positive bacteria (heliobacteria); and the acidobacteria.

Light reaction

The light reactions begin when exciton energy strikes the special pair of bacteriochlorophyll a molecule. The absorption of energy excites the special pair, converting it from a relatively weak to a very strong electron donor (very electronegative reduction potential). Once this strong donor has been produced, the remaining steps in electron flow simply conserve the energy released when electrons flow through a membrane from carriers of low E0` to those of high E0`, generating a proton motive force. (Please recall the electron transport chain).

Reducing power (NADH or NADPH) is also necessary so that CO2 can be reduced to the redox level of cell material. As previously mentioned, reducing power for purple sulfur bacteria comes from hydrogen sulfide (H2S). Using reverse electron flow, (using ATP to synthesize NADPH) these organisms synthesize NADPH.

Plant, cyanobacteria and algal photosynthesis (oxygenic photosynthesis)

In contrast to electron flow in anoxygenic phototrophs, electron flow in oxygenic phototrophs proceeds through two distinct but interconnected series of light reactions. The two light systems are called photosystem I and photosystem II, each photosystem having a spectrally distinct form of reaction center chlorophyll a. Photosystem I (PSI) chlorophyll, called P700, absorbs light at long wavelengths (far red light), whereas PSII chlorophyll, called P680, absorbs light at shorter wavelengths (near red light).

CO2 fixation in photoautotrophs

Either plant or bacterial photosynthesis, after ATP and NADPH are produced through light reactions (photophosphorylation), the energy and electrons were used for fixing of CO2 (autotrophy). There are three distinct pathways are available for fixing the CO2 in these systems.

1. Calvin cycle: The first step in the Calvin cycle is catalyzed by the enzyme ribulose bisphosphate carboxylase, RubisCO for short. The Calvin cycle is operative in purple bacteria, cyanobacteria, algae, green plants, most chemolithotrophic Bacteria, and even in a few Archaea. RubisCO catalyzes the formation of two molecules of 3-phosphoglyceric acid (PGA) from ribulose bisphosphate and CO2. The PGA is then phosphorylated and reduced to a key intermediate of glycolysis, glyceraldehyde 3-phosphate. From here, glucose can be formed by reversal of the early steps in glycolysis. 12 NADPH and 18 ATP are required to synthesize one C6 sugar (hexose) from 6 CO2 by the Calvin cycle.

2. Reverse TCA cycle

Green sulfur bacteria such as Chlorobium fix CO2 by a reversal of steps in the citric acid cycle, a pathway called the reverse citric acid cycle. This pathway requires the activity of two ferredoxin-linked enzymes that catalyze the reductive fixation of CO2; ferredoxin is produced in the light reactions of green sulfur bacteria.

3. Hydroxypropionate pathway

The green nonsulfur phototroph Chloroflexus grows autotrophically with either H2 or H2S as electron donor. However, neither the Calvin cycle nor the reverse citric acid cycle operates in this organism. Instead, two molecules of CO2 are reduced to glyoxylate by the hydroxypropionate pathway. This pathway is so named because hydroxypropionate, a three-carbon compound, is a key intermediate.

Chemo-lithotrophic-autotrophs

Organisms that obtain energy from the oxidation of inorganic compounds are called chemolithotrophs. Most chemolithotrophic bacteria are also autotrophs. As we have noted, for growth on CO2 as the sole carbon source an organism needs (1) ATP and (2) reducing power. Some chemolithotrophs grow as mixotrophs, meaning that although they conserve energy from the oxidation of an inorganic compound, they require an organic compound as their carbon source. ATP generation in chemolithotrophs is similar to that in chemoorganotrophs, except that the electron donor is inorganic rather than organic. The electrons from the inorganic source undergo electron transport, and ATP synthesis occurs by way of ATPases. Reducing power in chemolithotrophs is obtained in either of two ways: directly from the inorganic compound, if it has a sufficiently low reduction potential, such as H2, or by reverse electron transport reactions.

Hydrogen Oxidation: Hydrogen (H2) is a common product of microbial metabolism, and a number of chemolithotrophs are able to use it as an electron donor in energy metabolism. Many anaerobic H2-oxidizing Bacteria and Archaea are known, which differ in the electron acceptor they use (for example, nitrate, sulfate, ferric iron, and others). Here we consider the aerobic H2-oxidizing bacteria, organisms that couple the oxidation of H2 to the reduction of O2, forming water. Synthesis of ATP during H2 oxidation by O2 is the result of electron transport reactions that generate a proton motive force. The enzyme responsible for it is hydrogenase. Example: Hydrogenobacter thermophilus, Ralstonia eutropha.

Sulphur oxidation: Some of the bacteria oxidize Sulphur compounds (hydrogen sulphide, thiosulfate, Sulphur) into sulphite (SO3). The enzyme responsible is sulphite oxidase followed by sulfate formation (SO4). Sulfite oxidase transfers electrons from SO3 directly to cytochrome c, and ATP is made from this during subsequent electron transport and proton motive force formation. The example organism is Thiobacillus thiooxidans.

Iron oxidation: The aerobic oxidation of ferrous (Fe2+) to ferric (Fe3+) iron supports growth of the chemolithotrophic “iron bacteria.” At acidic pH, only a small amount of energy is available from this oxidation and for this reason the iron bacteria must oxidize large amounts of iron in order to grow. The ferric iron produced forms insoluble ferric hydroxide [Fe(OH)3] and otheriron precipitates in water that drive down the pH. This explains why most iron-oxidizing bacteria are obligately acidophilic. Example: Thiobacullus ferrooxidans.

Ammonia oxidation (or Nitrification): The inorganic nitrogen compounds ammonia (NH3) and nitrite (NO2-) are chemolithotrophic substrates and are oxidized aerobically by the “nitrifying bacteria” in the process of nitrification. Nitrifying bacteria are widely distributed in soils and water. One group (for example, Nitrosomonas) oxidizes NH3 to nitrite (NO2-), and another group (for example, Nitrobacter and Nitrospira) oxidizes NO2- to NO3-. The complete oxidation of NH3 to NO3-, an eight-electron transfer, is thus carried out by the concerted activity of two groups of organisms. The bioenergetics of nitrification is based on the same principles that govern other chemolithotrophic reactions: Electrons from reduced inorganic substrates (in this case, reduced nitrogen compounds) enter an electron transport chain, and electron flow establishes a proton motive force that drives ATP synthesis. The key enzymes are NH3 is oxidized by ammonia monooxygenase whereas the nitrite-oxidizing bacteria employ the enzyme nitrite oxidoreductase to oxidize NO2- to NO3-, with electrons traveling a very short electron transport chain.

Carbon fixation in chemolithotrophs: Sulfur- and iron-oxidizing chemolithotrophs and aerobic nitrifying bacteria employ the Calvin cycle for CO2 fixation.

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