Bacteriology at UW-Madison

The Microbial World

Lectures in Microbiology by Kenneth Todar PhD    University of Wisconsin-Madison    Department of Bacteriology

Diversity of Microbial Metabolism

Introduction

A lot of hoopla is made about microbial diversity. Although eucaryotic microbes, especially the protista, exhibit a great deal of structural diversity, the procaryotes are without this distinction. However, based on their modes of metabolism, the procaryotes are much more diverse than all eucaryotes, and the real real explanation for "microbial diversity" rests fundamentally on some aspect procaryotic metabolism, especially with regards to energy-generating metabolism and synthesis of secondary metabolites. Microbial diversity translates to metabolic diversity. The procaryotes, as a group, conduct all the same types of basic metabolism as eucaryotes, but, in addition, there are several types of energy-generating metabolism among the procaryotes that are non existent in eucaryotic cells or organisms. These include

Unique fermentation pathways that produce a wide array of end products

Anaerobic respiration: respiration that uses substances other than O₂ as a final electron acceptor

Lithotrophy: use of inorganic substances as sources of energy

Photoheterotrophy: use of organic compounds as a carbon source during bacterial photosynthesis

Anoxygenic photosynthesis: uses special chlorophylls and occurs in the absence of O₂

Methanogenesis: an ancient type of archaean metabolism that uses H₂ as an energy source and produces methane

Light-driven nonphotosynthetic energy production: unique archaean metabolism that converts light energy into chemical energy; occurs in the archaea (extreme halophiles)

Unique mechanisms for autotrophic CO₂ fixation,  including primary production on anaerobic habitats

Figure 2. The structure of ATP. ATP is derived from the nucleotide adenosine monophosphate (AMP) or adenylic acid, to which two additional phosphate groups are attached through pyrophosphate bonds (~P). These two bonds are energy rich in the sense that their hydrolysis yields a great deal more energy than a corresponding covalent bond. ATP acts as a coenzyme in energetic coupling reactions wherein one or both of the terminal phosphate groups is removed from the ATP molecule with the bond energy being used to transfer part of the ATP molecule to another molecule to activate its role in metabolism. For example, Glucose + ATP -----> Glucose-P + ADP  or  Amino Acid + ATP ----->AMP-Amino Acid + PPi.

Because of the central role of ATP in energy-generating metabolism, expect to see its involvement as a coenzyme in most energy-producing processes in cells.

NAD

Another coenzyme commonly involved in metabolism, derived from the vitamin niacin, is the pyridine nucleotide,  NAD (Nicotinamide Adenine Dinucleotide). The basis for chemical transformations of energy usually involves oxidation/reduction reactions. For a biochemical to become oxidized, electrons must be removed by an oxidizing agent. The oxidizing agent is an electron acceptor that becomes reduced in the reaction. During the reaction, the oxidizing agent is converted to a reducing agent that can add its electrons to another chemical, thereby reducing it, and reoxidizing itself. The molecule that usually functions as the electron carrier in these types of coupled oxidation-reduction reactions in biological systems is NAD and its phosphorylated derivative, NADP.  NAD or NADP can become alternately oxidized or reduced by the loss or gain of two electrons. The oxidized form of NAD is symbolized NAD; the reduced form is symbolized as  NADH₂. The structure of NAD is drawn below.

ATP Synthesis

Substrate level phosphorylation (SLP) is the simplest, oldest and  least-evolved way to make ATP. In a substrate level phosphorylation, ATP is made during the conversion of an organic molecule from one form to another. Energy released during the conversion is partially conserved during the synthesis of the high energy bond of ATP. SLP occurs during fermentations and respiration (the TCA cycle), and even during some lithotrophic transformations of inorganic substrates.

Figure 4. Three examples of substrate level phosphorylation. (a) and (b) are the two substrate level phosphorylations that occur during the Embden Meyerhof pathway, but they occur in all other fermentation pathways which have an Embden-Meyerhof component. (c) is a substrate level phosphorylation found in Clostridium and Bifidobacterium. These are two anaerobic (fermentative) bacteria who learned how to make one more ATP from glycolysis beyond the formation of pyruvate.

Electron Transport Phosphorylation (ETP) is a much more complicated affair that evolved long after SLP. Electron Transport Phosphorylation takes place during respiration, photosynthesis, lithotrophy and possibly other types of bacterial metabolism. ETP requires that electrons removed from substrates be dumped into an electron transport system (EST) contained within a membrane. The electrons are transferred through the EST to some final electron acceptor in the membrane (like O₂ in aerobic respiration) , while their traverse through the ETS results in the extrusion of protons and the establishment of a proton motive force (pmf) across the membrane. An essential component of the membrane for synthesis of ATP is a membrane-bound ATPase (ATP synthetase) enzyme. The ATPase enzyme transports protons, thereby utilizing the pmf (protons) during the synthesis of ATP. The idea in electron transport phosphorylation is to drive electrons through an ETS in the membrane, establish a pmf, and use the pmf to synthesize ATP. Obviously, ETP take a lot more "gear" than SLP, in the form of membranes, electron transport systems, ATPase enzymes, etc.

A familiar example of energy-producing and energy-consuming functions of the bacterial membrane, related to the establishment and use of pmf and the production of ATP, is given in the following drawing of the plasma membrane of Escherichia coli.

Figure 5. The plasma membrane of Escherichia coli. The membrane in cross-section reveals various transport systems, the flagellar motor apparatus (S and M rings), the respiratory electron transport system, and the membrane-bound ATPase enzyme. Reduced NADH + H+ feeds pairs of electrons into the ETS. The ETS is the sequence of electron carriers in the membrane [FAD --> FeS --> QH2 (Quinone) --> (cytochromes) b --> b --> o] that ultimately reduces O₂ to H₂O during respiration. At certain points in the electron transport process, the electrons pass "coupling sites" and this results in the translocation of protons from the inside to the outside of the membrane, thus establishing the proton motive force (pmf) on the membrane. The pmf is used in three ways by the bacterium to do work or conserve energy: active transport (e.g. lactose and proline symport; calcium and sodium antiport); motility (rotation of the bacterial flagellum), and ATP synthesis (via the ATPase enzyme during the process of oxidative phosphorylation or electron transport phosphorylation).

Heterotrophic Types of Metabolism

Fermentation

By definition, fermentation may be as simple as two steps illustrated in the following model. Indeed, some amino acid fermentations by the clostridia are this simple. But the pathways of fermentation are a bit more complex, usually involving several preliminary steps to prime the energy source for oxidation and substrate level phosphorylations.

Figure 6. Model fermentation. L. The substrate is oxidized to an organic intermediate; the usual oxidizing agent is NAD. Some of the energy released by the oxidation is conserved during the synthesis of ATP by the process of substrate level phosphorylation. Finally, the oxidized intermediate is reduced to end products. Note that NADH₂ is the reducing agent, thereby balancing its redox ability to drive the energy-producing reactions. R. In lactic fermentation by Lactobacillus, the substrate (glucose) is oxidized to pyruvate, and pyruvate becomes reduced to lactic acid. Redox balance is maintained by coupling oxidations to reductions within the pathway. For example, in lactic acid fermentation via the EmbdenMeyerhof pathway, the oxidation of glyceraldehyde phosphate to phosphoglyceric acid is coupled to the reduction of pyruvic acid to lactic acid.

In biochemistry, for the sake of convenience, fermentation pathways start with glucose. This is because it is the simplest molecule, requiring the fewest enzymatic ( catalytic) steps, to enter into a pathway of glycolysis and central metabolism.

In the bacteria there exist three major pathways of glycolysis (the dissimilation of sugars): the classic Embden-Meyerhof pathway, which is also used by most eucaryotes, including yeast (Saccharomyces): the heterolactic pathway used by lactic acid bacteria, and the Entner-Doudoroff pathway used by vibrios and pseudomonads, including Zymomonas. Although the latter two pathways have some interesting applications in the manufacture of dairy products and alcoholic beverages, they will not be discussed further in this section..

The Embden-Meyerhof Pathway

Figure 7. The Embden Meyerhof pathway for glucose dissimilation. The overall reaction is the oxidation of glucose to 2 pyruvic acid. The two branches of the pathway after the cleavage are identical.

The first three steps of the pathway prime (phosphorylated) and rearrange the hexes for cleavage into 2 triodes (glyceraldehyde phosphate). Fructose 1,6-diphosphate aldolase is the key (cleavage) enzyme in the E-M pathway. Each triose molecule is oxidized and phosphorylated followed by two substrate level phosphorylations that yield 4 ATP during the drive to pyruvate.

Lactic acid bacteria reduce the pyruvate to lactic acid; yeast reduce the pyruvate to alcohol (ethanol) and CO₂ as shown in Figure 8 below.

Figure 8. (a) The Embden Meyerhof pathway of lactic acid fermentation in lactic acid bacteria (Lactobacillus) and (b) the Embden Meyerhof pathway of alcohol fermentation in yeast (Saccharomyces). The pathways yield two moles of end products and two moles of ATP per mole of glucose fermented. The steps in the breakdown of glucose to pyruvate are identical. The difference between the pathways is the manner of reducing pyruvic acid, thereby giving rise to different end products.

Besides lactic acid, Embden-Meyerhof fermentations in bacteria can lead to a wide array of end products depending on the pathways taken in the reductive steps after the formation of pyruvic acid. Usually, these bacterial fermentations are distinguished by their end products into the following groups.

1. Homolactic Fermentation. Lactic acid is the sole end product. Pathway of the homolactic acid bacteria (Lactobacillus and most streptococci). The bacteria are used to ferment milk and milk products in the manufacture of yogurt, buttermilk, sour cream, cottage cheese, cheddar cheese, and most fermented dairy products.

2. Mixed Acid Fermentations. Mainly the pathway of the Enterobacteriaceae. End products are a mixture of lactic acid, acetic acid, formic acid, succinate and ethanol, with the possibility of gas formation (CO₂ and H₂) if the bacterium possesses the enzyme formate dehydrogenase, which cleaves formate to the gases.

2a. Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition, 2,3 butanediol from the condensation of 2 pyruvate. The use of the pathway decreases acid formation (butanediol is neutral) and causes the formation of a distinctive intermediate, acetoin. Water microbiologists have specific tests to detect low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella).

3. Butyric acid fermentations, as well as the butanol-acetone fermentation (below), are run by the clostridia, the masters of fermentation. In addition to butyric acid, the clostridia form acetic acid, CO₂ and H₂ from the fermentation of sugars. Small amounts of ethanol and isopropanol may also be formed.

3a. Butanol-acetone fermentation. Butanol and acetone were discovered as the main end products of fermentation by Clostridium acetobutylicum during the World War I. This discovery solved a critical problem of explosives manufacture (acetone is required in the manufacture gunpowder) and is said to have affected the outcome of the War. Acetone was distilled from the fermentation liquor of Clostridium acetobutylicum, which worked out pretty good if you were on our side, because organic chemists hadn't figured out how to synthesize it chemically. You can't run a war without gunpowder, at least you couldn't in those days.

4. Propionic acid fermentation. This is an unusual fermentation carried out by the propionic acid bacteria which include corynebacteria, Propionibacterium and Bifidobacterium. Although sugars can be fermented straight through to propionate, propionic acid bacteria will ferment lactate (the end product of lactic acid fermentation) to acetic acid, CO₂ and propionic acid. The formation of propionate is a complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate are converted to 2 moles of propionate + 1 mole of acetate + 1 mole of CO₂, and 1 mole of ATP is squeezed out in the process. The propionic acid bacteria are used in the manufacture of Swiss cheese, which is distinguished by the distinct flavor of propionate and acetate, and holes caused by entrapment of CO₂.

The Embden-Meyerhof pathway for glucose dissimilation (Figure 8), as well as the TCA cycle discussed below (Figure 10), are two pathways that are at the center of metabolism in nearly all organisms. Not only do these pathways dissimilate organic compounds and provide energy, they also provide the precursors for biosynthesis of macromolecules that make up living systems. These are sometimes called amphibolic pathways since the have both an anabolic and a catabolic function.

Respiration

1. The tricarboxylic acid (TCA) cycle (also known as the citric acid cycle or the Kreb's cycle): when an organic compound is utilized as a substrate, the TCA cycle is used for the complete oxidation of the substrate. The end product that always results from the complete oxidation of an organic compound is CO₂.

2. A membrane and an associated electron transport system (ETS). The ETS is a sequence of electron carriers in the plasma membrane that transports electrons taken from the substrate through the chain of carriers to a final electron acceptor. The electrons enter the ETS at a very low redox potential (E'_o) and exit at a relatively high redox potential. This drop in potential releases energy that can be harvested by the cells in the process of ATP synthesis by the mechanisms of electron transport phosphorylation. The operation of the ETS establishes a proton motive force (pmf) due to the formation of a proton gradient across the membrane.

3. An outside electron acceptor ("outside", meaning it is not internal to the pathway, as is pyruvate in a fermentation). For aerobic respiration the electron acceptor is O₂, of course. Molecular oxygen is reduced to H₂0 in the last step of the electron transport system. But in the bacterial processes of anaerobic respiration, the final electron acceptors may be SO₄ or S or NO₃ or NO₂ or certain other inorganic compounds, or even an organic compound, such as fumarate.

4. A transmembranous ATPase enzyme (ATP synthetase). This enzyme utilizes the proton motive force established on the membrane (by the operation of the ETS) to synthesize ATP in the process of electron transport phosphorylation. It is believed that the transmembranous Fo subunit is a proton transport system that transports 2H⁺ to the F1 subunit (the actual ATPase) on the inside of the membrane. The 2 protons are required and consumed during the synthesis of ATP from ADP plus Pi. See Figure 6 -the membrane of E. coli. The reaction catalyzed by the ATPase enzyme is ADP + Pi + 2 H⁺ <----------> ATP. (It is important to appreciate the reversibility of this reaction in order to account for how a fermentative bacterium, without an ETS, could establish a necessary pmf on the membrane for transport or flagellar rotation. If such an organism has a transmembranous ATPase, it could produce ATP by SLP, and subsequently the ATPase could hydrolyze the ATP, thereby releasing protons to the outside of the membrane.)

The diagram below of aerobic respiration (Figure 9) integrates these metabolic processes into a scheme that represents the overall process of respiratory metabolism. A substrate such as glucose is completely oxidized to to CO₂ by the combined pathways of glycolysis and the TCA cycle. Electrons removed from the glucose by NAD are fed into the ETS in the membrane. As the electrons traverse the ETS, a pmf becomes established across the membrane. The electrons eventually reduce an outside electron acceptor, O₂, and reduce it to H₂0. The pmf on the membrane is used by the ATPase enzyme to synthesize ATP by a process referred to as "oxidative phosphorylation".

Figure 9. Model of Aerobic respiration.

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The overall reaction for the aerobic respiration of glucose is

Biological methanogenesis is the source of methane (natural gas) on the planet. Methane is preserved as a fossil fuel (until we use it all up) because it is produced and stored under anaerobic conditions, and oxygen is needed to oxidize the CH₄ molecule. Methanogenesis is not really a form of anaerobic respiration, but it is a type of energy-generating metabolism that requires an outside electron acceptor in the form of CO₂.

Denitrification is an important process in agriculture because it removes NO₃ from the soil. NO₃ is a major source of nitrogen fertilizer in agriculture. Almost one-third the cost of some types of agriculture is in nitrate fertilizers The use of nitrate as a respiratory electron acceptor is usually an alternative to the use of oxygen. Therefore, soil bacteria such as Pseudomonas and Bacillus will use O₂ as an electron acceptor if it is available, and disregard NO₃. This is the rationale in maintaining well-aerated soils by the agricultural practices of plowing and tilling. E. coli will utilize NO₃ (as well as fumarate) as a respiratory electron acceptor and so it may be able to continue to respire in the anaerobic intestinal habitat.

Sulfate reduction is not an alternative to the use of O₂ as an electron acceptor. It is an obligatory process that occurs only under anaerobic conditions. Methanogens and sulfate reducers may share habitat, especially in the anaerobic sediments of eutrophic lakes such as Lake Mendota, where they crank out methane and hydrogen sulfide at a surprising rate.

Anaerobic respiring bacteria and methanogens play an essential role in the biological cycles of carbon, nitrogen and sulfur. In general, they convert oxidized forms of the elements to a more reduced state. The lithotrophic procaryotes metabolize the reduced forms of nitrogen and sulfur to a more oxidized state in order to produce energy. The methanotrophic bacteria, which uniquely posses the enzyme methane monooxygenase, can oxidize methane as a source of energy. Among all these groups of procaryotes there is a minicycle of the elements in a model ecosystem.

Lithotrophic Types of Metabolism

Some lithotrophs are facultative lithotrophs, meaning they are able to use organic compounds, as well, as sources of energy. Other lithotrophs do not use organic compounds as sources of energy; in fact, they won't transport organic compounds. CO₂ is the sole source of carbon for the methanogens and the nitrifying bacteria and a few other species scattered about in other groups.

Most lithotrophs get their carbon from from CO₂ and are thus autotrophs and are properly referred to as lithoautotrophs or chemoautotrophs. The lithotrophs are a very diverse group of procaryotes, united only by their ability to oxidize an inorganic compound as an energy source.

Lithotrophy runs through the Bacteria and the Archaea. If one considers methanogen oxidation of H₂ a form of lithotrophy, then probably most of the Archaea are lithotrophs. Lithotrophs are usually organized into "physiological groups" based on their inorganic substrate for energy production and growth (see Table 2 below).

The hydrogen bacteria oxidize H₂ (hydrogen gas) as an energy source. The hydrogen bacteria are facultative lithotrophs as evidenced by the pseudomonads that fortuitously possess a hydrogenase enzyme that will oxidize H₂ and put the electrons into their respiratory ETS. They will use H₂ if they find it in their environment even though they are typically heterotrophic. Indeed, most hydrogen bacteria are nutritionally versatile in their ability to use a wide range of carbon and energy sources.  the bacterial electron transport system.

The methanogens used to be considered a major group of hydrogen bacteria - until it was discovered that they are Archaea. The methanogens are able to oxidize H₂ as a sole source of energy while transferring the electrons from H₂ to CO₂ in its reduction to methane.  Metabolism of the methanogens is absolutely unique, yet methanogens represent the most prevalent and diverse group of Archaea. Methanogens use H₂ and CO₂ to produce cell material and methane. They have unique enzymes and electron transport processes. Their type of energy generating metabolism is never seen in the Bacteria, and their mechanism of autotrophic CO₂ fixation is very rare, except in methanogens.

The carboxydobacteria are able to oxidize CO (carbon monoxide) to CO₂, using an enzyme CODH (carbon monoxide dehydrogenase). The carboxydobacteria are not obligate CO users, i.e., some are also hydrogen bacteria, and some are phototrophic bacteria. Interestingly, the enzyme CODH used by the carboxydobacteria to oxidize CO to CO₂, is used by the methanogens for the reverse reaction - the reduction of CO₂ to CO - in their unique pathway of CO₂ fixation.

The nitrifying bacteria are represented by two genera, Nitrosomonas and Nitrobacter. Together these bacteria can accomplish the oxidation of NH₃ to NO₃, known as the process of nitrification. No single organism can carry out the whole oxidative process. Nitrosomonas oxidizes ammonia to NO₂ and Nitrobacter oxidizes NO₂ to NO₃. Most of the nitrifying bacteria are obligate lithoautotrophs, the exception being a few strains of Nitrobacter that will utilize acetate. CO₂ fixation utilizes RUBP carboxylase and the Calvin Cycle. Nitrifying bacteria grow in environments rich in ammonia, where extensive protein decomposition is taking place. Nitrification in soil and aquatic habitats is an essential part of the nitrogen cycle.

Lithotrophic sulfur oxidizers include both Bacteria (e.g. Thiobacillus) and Archaea (e.g. Sulfolobus). Sulfur oxidizers oxidize H₂S (sulfide) or S (elemental sulfur) as a source of energy. Similarly, the purple and green sulfur bacteria oxidize H₂S or S as an electron donor for photosynthesis, and use the electrons for CO₂ fixation (the dark reaction of photosynthesis). Obligate autotrophy, which is nearly universal among the nitrifiers, is variable among the sulfur oxidizers. Lithoautotrophic sulfur oxidizers are found in environments rich in H₂S, such as volcanic hot springs and fumaroles, and deep-sea thermal vents. Some are found as symbionts and endosymbionts of higher organisms. Since they can generate energy from an inorganic compound and fix CO₂ as autotrophs, they may play a fundamental role in primary production in environments that lack sunlight. As a result of their lithotrophic oxidations, these organisms produce sulfuric acid (SO₄), and therefore tend to acidify their own environments. Some of the sulfur oxidizers are acidophiles that will grow at a pH of 1 or less. Some are hyperthermophiles that grow at temperatures of 115 degrees C.

Iron bacteria oxidize Fe⁺⁺ (ferrous iron) to Fe⁺⁺⁺ (ferric iron). At least two bacteria probably oxidize Fe⁺⁺ as a source of energy and/or electrons and are capable of lithoautotrophic growth: the stalked bacterium Gallionella, which forms flocculant rust-colored colonies attached to objects in nature, and Thiobacillus ferrooxidans, which is also a sulfur-oxidizing lithotroph.

Figure 11. Lithotrophic oxidations. These reactions produce energy for metabolism in the nitrifying and sulfur oxidizing bacteria.

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Phototrophic Metabolism

Photosynthesis is the conversion of light energy into chemical energy that can be used in the formation of cellular material from CO₂. Photosynthesis is a type of metabolism separable into a catabolic and anabolic component. The catabolic component of photosynthesis is the light reaction, wherein light energy is transformed into electrical energy, then chemical energy. The anabolic component involves the fixation of CO₂ and its use as a carbon source for growth, usually called the dark reaction. In photosynthetic procaryotes there are two types of photosynthesis and two types of CO₂ fixation.

The Light Reactions depend upon the presence of chlorophyll, the primary light-harvesting pigment in the membrane of photosynthetic organisms. Absorption of a quantum of light by a chlorophyll molecule causes the displacement of an electron at the reaction center. The displaced electron is an energy source that is moved through a membrane photosynthetic electron transport system, being successively passed from an iron-sulfur protein (X ) to a quinone to a cytochrome and back to chlorophyll (Figure 12 below). As the electron is transported, a proton motive force is established on the membrane, and ATP is synthesized by an ATPase enzyme. This manner of converting light energy into chemical energy is called cyclic photophosphorylation.

Figure 12. Photosystem I: cyclical electron flow coupled to photophosphorylation

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There are several types of pigments distributed among various phototrophic organisms. Chlorophyll is the primary light-harvesting pigment in all photosynthetic organisms. Chlorophyll is a tetrapyrrole which contains magnesium at the center of the porphyrin ring. It contains a long hydrophobic side chain that associates with the photosynthetic membrane. Cyanobacteria have chlorophyll a, the same as plants and algae. The chlorophylls of the purple and green bacteria, called bacteriochlorophylls are chemically different than chlorophyll a in their substituent side chains. This is reflected in their light absorption spectra. Chlorophyll a absorbs light in two regions of the spectrum, one around 450nm and the other between 650 -750nm; bacterial chlorophylls absorb from 800-1000nm in the far red region of the spectrum.

The chlorophylls are partially responsible for light harvesting at the photochemical reaction center. The energy of a photon of light is absorbed by a special chlorophyll molecule at the reaction center, which becomes instantaneously oxidized by a nearby electron acceptor of low redox potential. The energy present in a photon of light is conserved as a separation of electrical charge which can be used to generate a proton gradient for ATP synthesis.

Carotenoids are always associated with the photosynthetic apparatus. They function as secondary light-harvesting pigments, absorbing light in the blue-green spectral region between 400-550 nm. Carotenoids transfer energy to chlorophyll, at near 100 percent efficiency, from wave lengths of light that are missed by chlorophyll. In addition, carotenoids have an indispensable function to protect the photosynthetic apparatus from photooxidative damage. Carotenoids have long hydrocarbon side chains in a conjugated double bond system. Carotenoids "quench" the powerful oxygen radical, singlet oxygen, which is invariably produced in reactions between chlorophyll and O₂ (molecular oxygen). Some nonphotosynthetic bacterial pathogens, i.e., Staphylococcus aureus, produce carotenoids that protect the cells from lethal oxidations by singlet oxygen in phagocytes.

Phycobiliproteins are the major light harvesting pigments of the cyanobacteria. They also occur in some groups of algae. They may be red or blue, absorbing light in the middle of the spectrum between 550 and 650nm. Phycobiliproteins consist of proteins that contain covalently-bound linear tetrapyrroles (phycobilins). They are contained in granules called phycobilisomes that are closely associated with the photosynthetic apparatus. Being closely linked to chlorophyll they can efficiently transfer light energy to chlorophyll at the reaction center.

Figure 17. The distribution of photosynthetic pigments among photosynthetic microorganisms.

All phototrophic bacteria are capable of performing cyclic photophosphorylation as described above and in Figure 16 and below in Figure 18. This universal mechanism of cyclic photophosphorylation is referred to as Photosystem I. Bacterial photosynthesis uses only Photosystem I (PSI), but the more evolved cyanobacteria, as well as algae and plants, have an additional light-harvesting system called Photosystem II (PSII). Photosystem II is used to reduce Photosystem I when electrons are withdrawn from PSI for CO₂ fixation. PSII transfers electrons from H₂O and produces O₂, as shown in Figure 20.

Most of the phototrophic procaryotes are autotrophs, which means that they are able to fix CO₂ as a sole source of carbon for growth. Just as the oxidation of organic material yields energy, electrons and CO₂, in order to build up CO₂ to the level of cell material (CH₂O), energy (ATP) and electrons (reducing power) are required. The overall reaction for the fixation of CO₂ in the Calvin cycle is CO₂ + 3ATP + 2NADPH₂ ----------> CH₂O + 2ADP + 2Pi + 2NADP. The light reactions operate to produce ATP to provide energy for the dark reactions of CO₂ fixation. The dark reactions also need reductant (electrons). Usually the provision of electrons is in some way connected to the light reactions. A model for coupling the light and dark reactions of photosynthesis is illustrated in Figure 21 below.

Figure 21. Model for coupling the light and dark reactions of photosynthesis.

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The differences between plant and bacterial photosynthesis are summarized in Table 3 below. Bacterial photosynthesis is an anoxygenic process. The external electron donor for bacterial photosynthesis is never H₂O, and therefore, purple and green bacteria never produce O₂ during photosynthesis. Furthermore, bacterial photosynthesis is usually inhibited by O₂ and takes place in microaerophilic and anaerobic environments. Bacterial chlorophylls use light at longer wave lengths not utilized in plant photosynthesis, and therefore they do not have to compete with oxygenic phototrophs for light. Bacteria use only cyclic photophosphorylation (Photosystem I) for ATP synthesis and lack a second photosystem.

Table 3. Differences between plant and bacterial photosynthesis
 

	plant photosynthesis	bacterial photosynthesis
organisms	plants, algae, cyanobacteria	purple and green bacteria
type of chlorophyll	chlorophyll a  absorbs 650-750nm	bacteriochlorophyll  absorbs 800-1000nm
Photosystem I (cyclic photophosphorylation)	present	present
Photosystem I (noncyclic photophosphorylation)	present	absent
Produces O2	yes	no
Photosynthetic electron donor	H2O	H2S, other sulfur compounds or  certain organic compounds

While photosynthesis is highly-evolved in the procaryotes, it apparently originated in the Bacteria and did not spread or evolve in Archaea. But the Archaea, in keeping with their unique ways, are not without representatives which can conduct a type of light-driven photophosphorylation. The extreme halophiles, archaea that live in natural environments such as the Dead Sea and the Great Salt Lake at very high salt concentration (as high as 25 percent NaCl) adapt to the high-salt environment by the development of "purple membrane", actually patches of light-harvesting pigment in the plasma membrane. The pigment is a type of rhodopsin called bacteriorhodopsin which reacts with light in a way that forms a proton gradient on the membrane allowing the synthesis of ATP. This is the only example in nature of non photosynthetic photophosphorylation. These organisms are heterotrophs that normally respire by aerobic means. The high concentration of NaCl in their environment limits the availability of O₂ for respiration so they are able to supplement their ATP-producing capacity by converting light energy into ATP using bacteriorhodopsin.

Autotrophic CO₂ fixation

RUBP carboxylase (ribulose bisphosphate carboxylase) uses ribulose bisphosphate (RUBP) and CO₂ as co-substrates. In a complicated reaction the CO₂ is "fixed" by addition to the RUBP, which is immediately cleaved into two molecules of 3-phosphoglyceric acid (PGA). The fixed CO₂ winds up in the -COO group of one of the PGA molecules. Actually, this is the reaction which initiates the Calvin cycle (Figure 22 below).

The Calvin cycle is concerned with the conversion of PGA to intermediates in glycolysis that can be used for biosynthesis, and with the regeneration of RUBP, the substrate that drives the cycle. After the initial fixation of CO₂, 2 PGA are reduced and combined to form hexose-phosphate by reactions which are essentially the reverse of the oxidative Embden-Meyerhof pathway. (Now is a good time to go back to Figure 8 and look at the E-M pathway for the location of PGA and glucose-phosphate). The hexose phosphate is converted to pentose-phosphate, which is phosphorylated to regenerate RUBP. An important function of the Calvin cycle is to provide the organic precursors for the biosynthesis of cell material. Intermediates must be constantly withdrawn from the Calvin cycle in order to make cell material. In this regard, the Calvin cycle is an anabolic pathway. The fixation of CO₂ to the level of glucose (C₆H₁₂O₆) requires 18 ATP and 12 NADPH₂.

Figure 22. The Calvin cycle and its relationship to the synthesis of cell materials.

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The methanogens, a very abundant group of procaryotes, use CO₂ as a source of carbon for growth, and as a final electron acceptor in an energy-producing process that produces methane. If a methanogen is fed labeled CO₂ as a sole form of carbon, 95 percent of the label winds up in methane and 5 percent winds up in cell material. The methanogens fix CO₂ by means of the enzyme CODH (carbon monoxide dehydrogenase) and the Acetyl CoA pathway (Figure 23 below). Methanogens predominate in anaerobic habitats including the deep sea with its volcanos, thermal vents and fumaroles, and hence they perform a significant amount of CO₂ fixation on the planet.

Figure 23. The CODH or acetyl CoA pathway of CO₂ fixation in the methanogens. The pathway of methanogenesis steadily reduces CO₂ to the methyl (CH₃) level, mediated by the coenzyme methanopterin (MP), related to folic acid. MP-CH₃ may be reduced to methane (not shown) or the MP may be replaced by a vitamin B₁₂-like molecule to enter the pathway of CO₂ fixation. The "B₁₂"-CH₃ is substrate for CO fixation mediated by the CODH. CODH reduces CO₂ to CO and adds the CO to "B12"CH3 to form acetyl-[CODH]. Coenzyme A (CoA) then replaces the CODH, resulting in the formation of Acetyl CoA, which is in the heart of biosynthetic metabolism. The net effect is the reduction of 2 CO₂ to Acetyl CoA.

Biosynthesis

Biosynthesis or intermediary metabolism is a topic of biochemistry, more so than microbiology. It will not be dealt with in detail here. The fundamental metabolic pathways of biosynthesis are similar in all organisms, in the same way that protein synthesis or DNA structure are similar in all organisms. When biosynthesis proceeds from central metabolism as drawn below, some of the main precursors for synthesis of procaryotic cell structures and components are as follows.

Polysaccharide capsules or inclusions are polymers of glucose and other sugars.

Cell wall peptidoglycan (NAG and NAM) is derived from glucose phosphate.

Amino acids for the manufacture of proteins have various sources, the most important of which are pyruvic acid, alpha ketoglutaric acid and oxalacetic acid.

Nucleotides (DNA and RNA) are synthesized from ribose phosphate. ATP and NAD are part of purine (nucleotide) metabolism.

Triose-phosphates are precursors of glycerol, and acetyl CoA is a main precursor of lipids for membranes

Vitamins and coenzymes are synthesized in various pathways that leave central metabolism.  In the example given in Figure 24, heme synthesis proceeds from the serine pathway, as well as from succinate in the TCA cycle.