MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 5. MICROBIAL METABOLISM

ENERGY GENERATION AND REDUCTION EQUIVALENTS IN PHOTOTROPHIC MICROORGANISMS

For Photosynthesis, the visible spectrum of sunlight is of primary importance, specifically the wavelength range from 400 to 700 nm. Ultraviolet rays carry excessive energy and therefore destroy organic molecules (which is why UV irradiation is used for disinfection), whereas infrared rays lack sufficient energy to drive Chemical Reactions and are almost immediately converted into heat.

Based on their mechanism, types of photosynthesis are divided into two categories:

1. Anoxygenic photosynthesis does not involve the release of molecular oxygen. This more ancient process is characteristic of anaerobic prokaryotes: purple Bacteria, green bacteria, heliobacteria, and aerobic anoxygenic phototrophs. A unique type of photosynthesis associated with carotenoids is found among archaea, specifically the halobacteria.

2. Oxygenic photosynthesis, in which molecular oxygen is released into the atmosphere, is characteristic of two groups of prokaryotic organisms—cyanobacteria and prochlorophytes—as well as all eukaryotes, including Algae and higher plants.

The ability of organisms to survive using light energy is due to the presence of specific photoreceptor molecules known as pigments. All Photosynthetic Pigments belong to two chemical classes:

- pigments based on a tetrapyrrole Structure (chlorophylls, bacteriochlorophylls, and phycobiliproteins) (Fig. 5.20, a);

- pigments based on long polyisoprenoid chains (carotenoids) (Fig. 5.20, b).

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Fig. 5.20. Chemical Structure of pigments in photosynthetic microorganisms:

a - bacteriochlorophyll; b - beta-carotene

Chlorophylls (Chl) are magnesium-porphyrin pigments consisting of four interconnected pyrrole rings that form a closed cylindrical structure. They contain a metal atom (magnesium) linked to the nitrogen atoms of the pyrrole rings via covalent and coordination bonds. These pigments are characteristic of organisms that perform oxygenic photosynthesis. The chlorophylls of prokaryotes that carry out anoxygenic photosynthesis are called bacteriochlorophylls (Bchls). Six types of bacteriochlorophylls (a, b, c, d, e, g) and Two Types of chlorophylls (a and b) have been identified. Chlorophylls and bacteriochlorophylls absorb light in the range of 650-1035 nm.

Phycobiliproteins are red and blue pigments consisting of a protein subunit and a prosthetic group known as phycobilins. Phycobilins contain four pyrrole rings, but unlike chlorophylls, they form an open chain structure and do not contain a metal. They are referred to as open-chain tetrapyrroles. Phycobiliproteins absorb photons in the range of 480-650 nm and act as light-harvesting molecules. Cyanobacteria typically contain phycobiliproteins such as phycoerythrin, phycocyanin, and allophycocyanin.

Carotenoids are a large group of chemical compounds formed by the Condensation of typically eight isoprene units. Structurally, they can be aliphatic (lacking cyclic groups) or cyclic. If a molecule contains an aromatic ring at one end of the chain, the carotenoid is termed monocyclic; if at both ends, bicyclic. Carotenoids containing oxygen are classified as xanthophylls. Carotenoids absorb light in the 400-550 nm range and protect The Cell from damaging photooxidation reactions.

Based on their Functions, cellular pigments are divided into primary and accessory. Primary pigments comprise that fraction of bacteriochlorophylls (and chlorophylls) that are incorporated into the photochemical reaction center and drive the photochemical reaction. This function is performed by a fraction of Chl a and Bchl a, b, g. These represent a minor proportion of the total cellular pigment content: in anoxygenic phototrophs, primary bacteriochlorophyll accounts for 1/25 to 1/50 of the total, whereas in cyanobacteria, chlorophyll accounts for 1/30 to 1/100. In halobacteria, the primary pigments driving the photochemical reaction are carotenoids. Accessory pigments in photosynthetic microorganisms perform the following functions:

- absorb light quanta and transfer them to the photoreaction center, acting as antennae;

- participate in phototaxis;

- protect Cells against photooxidation.

Accessory pigments include a significant portion of Chl a, Bchl a, b, g, as well as other chlorophylls (bacteriochlorophylls), carotenoids (except in halobacteria), and phycobiliproteins (in cyanobacteria).

Unlike eukaryotes, prokaryotes lack specialized Organelles for photosynthesis (METABOLISM/14.html">Chloroplasts). In purple bacteria, the photosynthetic apparatus is localized in the cytoplasmic membrane and intracytoplasmic membranes; in green bacteria, in the cytoplasmic membrane and specialized structures called chlorosomes; and in cyanobacteria, in the cytoplasmic membrane and phycobilisomes, which are structurally similar to chlorosomes.

The photosynthetic apparatus of microorganisms consists of three main components:

1. Light-harvesting antennae, which absorb light energy and transfer it to photochemical reaction centers. They are also referred to as photoreceptor, light-harvesting, or antenna molecules.

2. Photochemical reaction centers (RC), where the electromagnetic energy is transformed into chemical energy.

3. Photosynthetic electron transport systems, which drive electron transfer coupled with ATP synthesis.

Light-harvesting antennas function to absorb photons and convert their energy into the energy of the singlet-excited state of BChl. Purple bacteria possess two types of Light-Harvesting Complexes (light-harvesting antennas):

- an inner (core) light-harvesting complex, which is always closely associated with the reaction center and surrounds it in a ring;

- a peripheral light-harvesting complex, located outside the inner complex and surrounding it. There may be one or several such complexes, and their protein content increases as light intensity decreases.

Light-harvesting antennas appear as oligomeric pigment-protein structures localized in the intracytoplasmic membranes. Carotenoid and BChl molecules are non-covalently bound to integral Membrane Proteins in such a way that their tetrapyrrole rings are held in a strictly fixed position relative to one another and to the membrane plane. The pigments that make up the antenna absorb light energy and transition to an excited singlet state. This Energy is transferred to other pigment molecules until it reaches the reaction center. Part of the energy is lost as heat or fluorescence. In green bacteria, light-harvesting antennas are housed in chlorosomes (Fig. 5.21)—elongated, lipid-rich vesicles located in the Cytoplasm and connected to the cytoplasmic membrane via a basal plate of crystalline structure. The interior of the chlorosome is packed with bundles of rod-like structures containing aggregates of BChl c, d, or e. These BChl molecules are not bound to proteins. Chlorosomes also contain Lipids and carotenoids. BChl a is localized in the basal plate, where it mediates The transfer of excitation energy from the chlorosomes to the reaction center.

Fig. 5.21. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF a chlorosome in green bacteria

Photochemical reaction centers (RCs). Two types of reaction centers have been identified in phototrophic organisms: RC I and RC II:

1. RC I is present in green sulfur bacteria (e.g., Chlorobium limicola, Prosthecochlorum aestuarii), heliobacteria (e.g., Heliobacterium chlorum, Heliobacillus mobilis), cyanobacteria, algae, and higher plants.

2. RC II is found in sulfur and non-sulfur purple bacteria, green non-sulfur bacteria, cyanobacteria, and phototrophic eukaryotes.

Reaction Center I. A special pair of BChl molecules serves as the primary electron donor (a BChl a dimer in green bacteria, and a BChl g dimer in heliobacteria). The redox potential (RP) of these Donors is +240 mV. Upon receiving energy from the excited BChl of the light-harvesting antennas, the primary donor transitions to an excited state. An electron from this strongly reduced primary donor is transferred to the primary acceptor—a monomeric BChl (Chl a isomer)—and subsequently to quinone A, an iron-sulfur protein [4Fe-4S], and quinone B.

Reaction Center II (quinone type). A special pair of BChl a or BChl b molecules acts as the primary electron donor, which becomes excited by energy absorbed and transferred from the light-harvesting antennas. An electron from the strongly reduced primary donor is transferred to the primary acceptor, which is an accessory BChl a, and then to bacteriopheophytin (BPh), followed by quinone A and quinone B.

Thus, reaction centers mediate the transformation of light energy into the energy of charge Separation (Membrane Potential – Δφ and the difference in redox potentials across the membrane – ΔEh). Charge separation occurs between the primary electron donor (a Chl or BChl dimer) located on the outer surface of the cytoplasmic membrane and the secondary electron acceptor (Quinones in RC II or iron-sulfur proteins in RC I).

Photosynthetic electron transport systems drive the subsequent transfer of the electron released from the primary donor through a chain of carriers. This transport establishes the electrochemical proton potential Δμ_Η+. The electron transport system includes the cytochrome b_C1 complex and cytochrome c₂. The cytochrome b_C1 complex functions as a proton pump, generating Δμ_Η+. Reduced cytochrome C₂ then transfers electrons back to the primary electron donor of the RC.

Mechanism of anoxygenic photosynthesis. Light-harvesting antennas, composed of BChl and carotenoids, absorb light energy in the 400–1100 nm range and transfer it to the photochemical reaction center (type I or type II — RC I or RC II).

In the dark, the bacteriochlorophyll molecule in the RC exists in a stable, unexcited state, with its electrons occupying the ground energy level. The Redox Potential of the reaction center is approximately +500 mV. When a light quantum reaches the bacteriochlorophyll molecule via the light-harvesting antennas, its electron absorbs a quantum of energy and jumps to a higher energy level, placing the bacteriochlorophyll molecule in an excited state. Concurrently, the redox potential of the excited reaction center drops to about -700 mV. In other words, upon absorbing a light quantum, the redox potential of the reaction center decreases by nearly 1200 mV, and the bacteriochlorophyll molecule becomes a potent reductant, serving as the primary electron donor. The bacteriochlorophyll molecule remains in this excited state for a very brief period (10^-9–10^-13 s).

Upon leaving the excited primary donor, the electron is transferred to the primary acceptor, then to the secondary acceptor, and continues along the photosynthetic Electron Transport Chain toward the terminal acceptor.

Electron flow can proceed in two directions:

1. Back to the bacteriochlorophyll molecule from which it originated (Fig. 5.22, 1). This movement is termed Cyclic electron transport. Cyclic electron transfer results in proton translocation across the cytoplasmic membrane and The formation of a proton gradient, which drives ATP synthesis. This Mechanism of ATP generation is called Photophosphorylation.

2. The electron does not return to the bacteriochlorophyll, but instead is transferred to NAD⁺ or oxidized ferredoxin (Fd⁺), which are subsequently used as reducing equivalents in anabolic reactions (Fig. 5.22, 2). Thus, the electron that left the bacteriochlorophyll molecule is removed from the "system." This creates an open-ended electron flow known as non-cyclic transport.

Photosynthetic bacteria may exhibit either exclusively cyclic transport (Purple and green filamentous bacteria) or the simultaneous functioning of both types of electron transfer. Regardless of the pathway taken by the electron ejected from bacteriochlorophyll, the cell experiences an electron deficit. In cyclic transport, There is a shortage of electrons for generating reducing equivalents, whereas in non-cyclic transport, electrons are needed to fill the "vacancy" left in the bacteriochlorophyll. Consequently, phototrophic organisms require additional electron sources—exogenous donors. These exogenous electron donors can be Organic compounds (in photoorganotrophs) or Inorganic Compounds (in photolithotrophs). Inorganic exogenous electron donors include reduced sulfur compounds (H₂S, SO₃^2-, thiosulfate, tetrathionate, thioglycolate) as well as molecular hydrogen.

Electrons released during The oxidation of exogenous donors are utilized by the cell for:

a) filling the electron "vacancy" in bacteriochlorophyll;

b) reducing NAD⁺.

The redox potentials of all potential exogenous donors are low enough to transfer an electron to oxidized bacteriochlorophyll without energy input. This is achieved by electron transfer along the electron transport chain down the electrochemical gradient (from carriers with lower redox potentials to those with higher ones) (Fig. 5.22, 3). When electrons are transported from an exogenous donor to NAD⁺, the process can occur in two ways:

1. If molecular hydrogen with a redox potential of -420 mV serves as the electron donor, NAD⁺ reduction proceeds without Energy Expenditure (Fig. 5.22, 4).

2. If the electron donors are reduced sulfur compounds or succinate, whose redox potentials are significantly higher than that of the NAD⁺/NADH₂ couple, electrons are transferred along the electron transport chain via reverse transport against the electrochemical gradient, requiring energy consumption (Fig. 5.22, 5).

Fig. 5.22. General scheme of electron transfer in anoxygenic photosynthesis:

RC - reaction center; RC* - reaction center with excited bacteriochlorophyll; Bph - bacteriopheophytin; Q - ubiquinone; b_C1, c - Cytochromes; 1-5 - pathways of Electron transfer from bacteriochlorophyll and exogenous donors

Purple bacteria are a morphologically diverse group of Gram-negative prokaryotes comprising over 50 species that inhabit freshwater bodies and marine sediments and are capable of anoxygenic photosynthesis. Purple bacteria contain bacteriochlorophylls a or b and over 50 different carotenoids (e.g., spirilloxanthin, rhodoxanthin, rhodopinal, spheroidene). Species containing bacteriochlorophyll a absorb sunlight with wavelengths up to 950 nm. In species with bacteriochlorophyll b, the absorption maximum in the red region of the spectrum is shifted toward longer wavelengths by more than 100 nm, reaching 1020–1030 nm. Thanks to their carotenoids, purple bacteria can also utilize shorter wavelengths of 400–550 nm. The combination of bacteriochlorophyll b and carotenoids allows their cells to grow across a very wide range of light wavelengths and, consequently, in numerous ecological niches inaccessible to other phototrophs. The quantity and composition of carotenoids determine the color of purple bacteria, which can be pink, red, orange, purple, brown, or yellowish-green. Pigment synthesis depends on culture age, nutrient medium composition, light intensity, and oxygen availability. Pigments are synthesized most efficiently under moderate illumination in anaerobic conditions, which are also optimal for photosynthesis.

In purple bacteria, the photosynthetic apparatus is localized in the cytoplasmic membrane and intracytoplasmic membranes.

Their exogenous electron donors can be either organic (succinate, fructose) or inorganic compounds (sulfide and other reduced sulfur compounds, molecular hydrogen, reduced iron). The Oxidation of reduced sulfur compounds proceeds via an intermediate product—elemental sulfur: H₂S → S⁰ → SO₄^2-. Depending on the localization of elemental sulfur, purple bacteria are subdivided into sulfur and non-sulfur species (Fig. 5.23). Purple sulfur bacteria are tolerant to high sulfide concentrations, whereas non-sulfur bacteria are sensitive.

Fig. 5.23. Systematics of purple bacteria

In terms of their type of metabolism, purple bacteria are the most exotic group of prokaryotes. They can grow photolithoautotrophically, photoorganoheterotrophically, chemolithoautotrophically, and chemoorganoheterotrophically. In some cases, individual species are capable of all four metabolic types. Table 5.6 lists the possible metabolic types of the non-sulfur purple bacterium Rhodospirillum rubrum under anaerobic conditions.

Table 5.6. Conditions supporting the growth of Rhodospirillum rubrum

For purple sulfur bacteria, photolithoautotrophy is the primary mode of existence. All representatives of this group can grow under illumination in anaerobic conditions on media containing CO₂ as the sole carbon source, utilizing reduced sulfur compounds or molecular hydrogen as exogenous electron donors. Organic substances can serve merely as additional carbon sources or growth factors, and only rarely as electron donors. All species can photometabolize acetate and Pyruvate, and only a few are capable of purely photoorganoheterotrophic growth. It was previously thought that purple sulfur bacteria are extremely sensitive to oxygen; however, it is now known that certain species are aerotolerant and even capable of chemolithotrophic growth at low oxygen concentrations, utilizing oxygen as a terminal electron acceptor. Aerobic chemoorganoheterotrophic growth has been proven for some species of Ectothiorhodospira and Thiocapsa. Certain purple sulfur bacteria exhibit The ability to grow in the dark under anaerobic conditions by fermenting certain sugars or organic acids.

Purple non-sulfur bacteria exhibit a strong preference for a photoorganoheterotrophic lifestyle, utilizing Fatty acids, alcohols, sugars, and Amino Acids as electron and carbon sources. Many species can also grow photolithoautotrophically, using molecular hydrogen and occasionally reduced sulfur compounds as electron donors. Some representatives of this group grow in the dark under microaerophilic or aerobic conditions, obtaining energy via aerobic Respiration. Their Krebs cycle, Glycolysis, and other chemoorganotrophic metabolic pathways function actively. The ability to grow anaerobically via nitrate respiration or Fermentation coupled with the oxidation of organic compounds has been discovered in members of the genus Rhodobacter. Thus, purple non-sulfur bacteria possess a diverse array of energy-generating mechanisms, including photosynthesis, aerobic and Anaerobic respiration, and fermentation.

When purple non-sulfur bacteria grow aerobically in the dark, their membranes resemble those typical of Gram-negative organisms. The cells are colorless or yellowish and contain very few photosynthetic pigments. Anaerobic conditions and illumination trigger drastic changes in their cellular and Membrane Structure: invaginations begin to form, leading to The Development of intracytoplasmic membranes that harbor the photosynthetic apparatus.

Green bacteria constitute a small group of microorganisms that perform anoxygenic photosynthesis. Their photosynthetic apparatus is located in the cytoplasmic membrane and specialized structures known as chlorosomes. These microorganisms are divided into two subgroups: green sulfur bacteria and green non-sulfur (filamentous) bacteria (Fig. 5.24, Table 5.7).

Fig. 5.24. Systematics of green bacteria

Green sulfur bacteria are Gram-negative, non-motile unicellular forms, obligate anaerobes, and obligate photolithotrophs. They utilize CO₂ as their carbon source. Green sulfur bacteria can assimilate certain organic compounds (a limited range of sugars, organic acids, and amino acids) as supplementary carbon sources, but never as primary carbon sources or exogenous electron donors. Their exogenous electron donors include H₂S, S⁰, Na₂S₂O₃, and H₂. During sulfide oxidation, which takes place in the periplasm, elemental sulfur is initially formed and always accumulates extracellularly. Once H₂S is depleted from the medium, S⁰ is taken up by the cells and oxidized to sulfate within the periplasmic space. Thus, green sulfur bacteria grow photolithoautotrophically. They contain bacteriochlorophylls c, d, and e along with trace amounts of bacteriochlorophyll a.

Table 5.7. Principal morphological, physiological, and biochemical differences between green sulfur and green non-sulfur (filamentous) bacteria

Green sulfur bacteria inhabit narrow ecological niches: illuminated anaerobic Water layers of lakes or coastal sediments.

Green non-sulfur (filamentous) bacteria consist of multiple rod-shaped cells, often enclosed within a mucilaginous sheath. Their Cell wall is Gram-negative yet flexible rather than rigid, enabling gliding motility. This group includes both mesophiles and thermophiles. Green non-sulfur bacteria are facultative anaerobes and phototrophs with a preference for organic exogenous electron donors. For instance, Chloroflexus aurantiacus grows in the light under both aerobic and anaerobic conditions in the presence of sugars, alcohols, organic acids, and amino acids. Certain strains of this species are capable of anaerobic photolithoautotrophic growth using H₂ or H₂S as electron donors. The oxidation of H₂S yields molecular sulfur, which is deposited in the medium as an amorphous mass and subsequently oxidized to sulfate in very small amounts. Chemoorganoheterotrophic growth is also possible under aerobic conditions, and in some strains, under anaerobic conditions as well. Green non-sulfur bacteria contain bacteriochlorophylls c or d, with their antenna molecules localized in chlorosomes.

Heliobacteria are obligately anaerobic phototrophic bacteria that perform anoxygenic photosynthesis and contain a unique bacteriochlorophyll g, which is absent in all other phototrophs. Four genera of heliobacteria are currently described: Heliobacterium (H. chlorum, H. gestii, H. sulfidophilum, H. undosum, H. modesticaldum), Heliobacillus (H. mobilis), Heliophilum (H. fasciatum), and Heliorestis (H. daurensis, H. baculata). Structurally, bacteriochlorophyll g resembles both chlorophyll a of Higher Plants and bacteriochlorophylls of purple and green bacteria. The absorption maximum of bacteriochlorophyll g is located at 788 nm (near the infrared region). Heliobacterial Cells also contain trace amounts of specialized C₃₀ carotenoids (other phototrophs contain C₄₀ derivatives). Another feature distinguishing heliobacteria from prokaryotic phototrophs is the absence of intracytoplasmic photosynthetic membranes (found in purple bacteria) and chlorosomes (found in green bacteria). Their pigments are localized exclusively in the cytoplasmic membrane. The primary electron donor in heliobacteria is bacteriochlorophyll g, and the primary acceptor is a specialized form of chlorophyll (not bacteriochlorophyll) a called hydroxychlorophyll a. This indicates a phylogenetic link between heliobacteria and oxygenic phototrophs. Heliobacteria grow exclusively under anaerobic conditions but exhibit both phototrophic and chemotrophic metabolism. Photoheterotrophic growth occurs in the light in the presence of certain organic carbon sources: pyruvate, lactate, acetate, or butyrate with CO₂, or ethanol with CO₂. Photoautotrophic metabolism has not been detected in any heliobacterial species due to the absence of known CO₂ fixation pathways. Chemo-

Heterotrophic metabolism in heliobacteria is carried out solely through pyruvate fermentation. Other organic compounds fail to support their chemoheterotrophic growth. Respiration is entirely absent in heliobacteria. Some species have been found capable of Nitrogen Fixation. Heliobacteria inhabit normal and flooded soils, and have been isolated from hot spring microbial mats and soda lakes, but never from open water or bottom sediments. Heliobacterium modesticaldum is a thermophilic species, whereas Heliorestis daurensis and Heliorestis baculata are alkaliphilic (growing at pH 9.0).

Aerobic anoxygenic phototrophs have been isolated from aerobic marine sands and cyanobacterial mats. This group includes mesophilic, thermophilic, halotolerant, and alkaliphilic species. Currently, aerobic anoxygenic bacteria are classified into eight genera. Two of them—Erythrobacter and Roseobacter—were isolated from marine environments, while six—Erythromicrobium, Roseococcus, Porphyrobacter, Acidiphilium, Erythromonas, and Sandaracinobacter—originate from freshwater niches. Their Morphology is quite diverse: Erythrobacter and Sandaracinobacter are typical slender rods capable of forming chains; the genus Roseococcus comprises cocci; and Porphyrobacter and Erythromonas are coccoid rods.

Most species of aerobic anoxygenic phototrophs are motile, possessing 1–3 flagella. A novel strain (JF-1) isolated from deep-sea hydrothermal vent water exhibits pleomorphic cells. Depending on age and medium composition, the cells may appear as cocci, bean-shaped structures, or filaments composed of several cells.

The pigments of aerobic anoxygenic phototrophs consist of A large number (around 20) of unusual carotenoids and bacteriochlorophyll. The carotenoids determine the red-pink-orange coloration of the cells and exhibit absorption peaks in the 420–550 nm range. Most species are dominated by bicyclic carotenoids, namely zeaxanthin and β-carotene. These bacteria contain only a single type of bacteriochlorophyll—Bchl a—at concentrations 10 to 20 times lower than those found in anaerobic phototrophs. The absorption maximum of protein-bound Bchl a lies within the 800–870 nm range. The ratio of carotenoids to Bchl a in the cell ranges from 1:8 to 1:10, respectively.

In cells of aerobic anoxygenic phototrophs, pigments are localized primarily in the cytoplasmic membrane. Intracytoplasmic membranes begin to form only under conditions of decreased oxygen partial pressure and environmental illumination. Carotenoids and Bchl a function as light-harvesting antennas, whereas the photoreaction center incorporates Bchl a and tetraheme c-type cytochromes with low- and high-potential Hemes. The electron transport system contains various ubiquinones (Q₈, Q₉, Q₁₀) and menaquinones (MK₈ or MK₉), albeit in smaller amounts than in purple bacteria. Various c-, b-, and a-type cytochromes are also present. Aerobic anoxygenic phototrophs can perform photophosphorylation only in the presence of molecular oxygen.

These bacteria possess a labile heterotrophic metabolism and are capable of oxidizing a range of organic substrates, including sugars, fatty acids, and amino acids. They contain active Enzymes of the Entner–Doudoroff pathway, the Krebs cycle, and The Glyoxylate cycle. Several species possess the enzymes of The Calvin Cycle and are capable of autotrophic growth. The presence of light stimulates CO₂ assimilation. These bacteria accumulate reserve Materials within their cells in the form of glycogen and polyphosphates.

The photosynthetic activity of aerobic phototrophic bacteria is not their primary energy source; nevertheless, it makes a certain contribution to generating the Electrochemical Potential across the cytoplasmic membrane.

A specific mechanism of anoxygenic photosynthesis in halobacteria.

Halophiles are microorganisms that inhabit hypersaline environments. Slight halophiles tolerate NaCl concentrations up to 5%, moderate halophiles up to 20%, and extreme halophiles up to 30%. High osmotic pressure in saline environments causes water efflux from the cell and Protein Denaturation. Therefore, halophiles comprise organisms capable of balancing the environmental osmotic pressure and possessing mechanisms to protect against the denaturing effects of salts. Microbial adaptation to high salt concentrations (osmoregulation) can be achieved in two ways:

1. Osmoregulation via compatible organic osmolytes. Osmotic equilibrium within the cell is maintained by accumulating small, highly water-soluble molecules in the cytoplasm, such as glycerol, arabitol, sugars and their derivatives, Amino Acids and their derivatives, betaine, ectoine, etc. This accumulation can occur both through Biosynthesis and by uptake of these compounds from the medium. The intracellular concentration of compatible organic osmolytes depends on the environmental salt concentration. The structure and activity of enzymes in microorganisms utilizing this type of osmoregulation are independent of the intracellular concentration of these compounds. Osmoregulation via compatible organic osmolytes is characteristic of most halophilic and halotolerant organisms.

2. Osmoregulation via maintaining a high intracellular salt concentration is achieved by the cell actively pumping NaCl outward while accumulating KCl inside. The intracellular KCl concentration is maintained at a level comparable to the environmental NaCl concentration (approximately 4 M), meaning halotolerance is ensured by establishing a Na⁺ and K⁺ concentration gradient. With this type of adaptation, cellular enzymes and structural components are adapted to high salt concentrations (proteins are rich in acidic amino acids; enzyme conformation and activity are maintained by high salt concentrations). This type of osmoregulation is characteristic exclusively of anaerobic halophilic fermenters of the order Haloanaerobiales and aerobic extremely halophilic archaea of the order Halobacteriales.

Aerobic extremely halophilic archaea (order Halobacteriales) derive additional energy from atypical photochemical reactions. These bacteria exhibit an aerobic chemoorganotrophic metabolism. They oxidize glucose via the Entner–Doudoroff pathway, generating proton-motive force primarily through respiration. However, because maintaining cellular osmotic balance demands high energy expenditures, and oxygen solubility in saline water is very low, halobacteria possess an additional mechanism for ATP generation driven by light.

The surface of halobacteria consists of red and purple membranes. Red membranes contain a large amount of carotenoids, which perform a photoreparative function (repairing thymine dimers induced by ultraviolet radiation). A typical Respiratory Chain with three phosphorylation sites is localized in the red membranes, where respiration takes place.

Purple membranes occupy approximately 50% of the halobacterial surface. They contain up to 75% rhodopsins and participate in photosynthesis. Four types of rhodopsins are known in archaea: two of them—Bacteriorhodopsin and halorhodopsin—function as ion pumps, whereas the other two act as photoreceptors in phototaxis.

Bacteriorhodopsin consists of the bacterioopsin protein and a retinal chromophore covalently bound to this protein. Retinal, in turn, is a derivative of β-carotene (a carotenoid) and is formed via its oxidative Cleavage in the presence of molecular oxygen. It is retinal that imparts the characteristic purple color to the membrane. Light is absorbed by the purple membranes. Upon Light absorption, retinal releases a proton (transitioning into a deprotonated state) and becomes bleached. The proton is released into the environment, establishing a hydrogen ion electrochemical potential gradient (Δμ_H+) that drives ATP synthesis. The deprotonated retinal of bacteriorhodopsin is reprotonated using a proton derived from the cytoplasm. Thus, retinal acts as a proton pump, extruding protons out of the cell.

Halorhodopsin is also a retinal-containing protein. It is believed that this compound is more widespread among halobacteria than bacteriorhodopsin. Upon light absorption, halorhodopsin transports chloride ions (Cl^-) into the cell. Since chloride ions carry a negative charge, importing chloride into the cell is energetically equivalent to exporting

protons outward. In this manner, halobacteria generate a chloride ion gradient, which also serves as an energy source.

Unlike classical photosynthesis, where ATP is generated via cyclic electron flow through chlorophyll, in halobacteria the transmembrane potential is established with the aid of carotenoids.

Fig. 5.25 illustrates the potential ion transport pathways in aerobic halophilic archaea. As shown in the figure, the proton gradient in halobacteria is generated in three ways: via aerobic respiration across red membranes (Fig. 5.25, 1); via light-driven proton translocation through bacteriorhodopsin in purple membranes (Fig. 5.25, 2); and, in the event of insufficient protons in the periplasm, via ATP Hydrolysis at ATP synthase using ATP produced through Substrate-Level Phosphorylation Reactions (Fig. 5.25, 3). The hydrogen ion electrochemical potential gradient (ΔμH+) established by the aforementioned mechanisms is utilized by halobacteria to synthesize ATP via ATP synthase (Fig. 5.25, 3) and to export Na⁺ cations from the cell, which is essential for osmoregulation (Fig. 5.25, 4). High intracellular K⁺ concentration is maintained via uniport (Fig. 5.25, 6). Cl^- enters the cell through two pathways: symport with Na⁺ (Fig. 5.25, 7) and photochemical processes mediated by halorhodopsin (Fig. 5.25, 8).

Fig. 5.25. Ion transport in aerobic halophilic archaea:

1 – proton extrusion during respiration; 2 – light-driven proton extrusion via bacteriorhodopsin; 3 – ATP synthesis driven by the proton gradient at ATP synthase; 4 – electrogenic sodium/proton antiport; 5 – Amino Acid Transport driven by the sodium gradient; 6 – potassium uniport driven by membrane potential; 7 – light-independent chloride transport system, possibly coupled with sodium influx; 8 – light-driven chloride ion import via halorhodopsin

Mechanism of oxygenic photosynthesis. Because sources of reduced sulfur compounds and molecular hydrogen in nature are quite limited, a major evolutionary step was the utilization of water by organisms as a novel exogenous electron donor. However, the redox potential of the H₂O/O₂ system is +810 mV; therefore, the electron "vacancy" in the reaction center bacteriochlorophyll molecule arising during noncyclic electron transport cannot be directly filled by an electron from a water molecule. For this to become possible, it is necessary, first, to "extract" electrons from the thermodynamically stable H₂O molecule and, second, to "elevate" them to a higher energy level enabling them to feed into the photosystem described above. This problem was resolved by the evolution of an additional pigment system—Photosystem II. It is associated with The Emergence of new pigments: chlorophylls a and b, and phycobiliproteins. It has been established that the reaction center chlorophyll a of photosystem II in the photo-oxidized state exhibits a redox potential of +1000 to +1300 mV, which is sufficiently positive to be reduced by electrons derived from water molecules. The Mechanism of the reactions coupled with electron transfer from water molecules to the P680 pigment remains unknown. This process has been shown to be stepwise, with manganese being an essential component of the system. Thus, photosystem II evolved as an adjunct to Photosystem I to enable The Use of water as an exogenous electron donor. Molecular oxygen is a byproduct of this process (Fig. 5.26).

Fig. 5.26. General scheme of electron transfer in oxygenic photosynthesis

The overall scheme of cyanobacterial photosynthesis represents a specific series of Reactions Involving Two consecutive photoreactions. Light is absorbed by the photoreceptors of photosystem II (phycobiliproteins, chlorophyll a, carotenoids) and transferred to the reaction center chlorophyll. An electron is detached from the chlorophyll and passed to plastoquinone, while the oxidized pigment molecule is reduced by electrons derived from water. The electron from the photosystem II acceptor travels through a chain of carriers to the photosystem I reaction center and reaches the photooxidized form of chlorophyll a—the P₇₀₀ pigment (redox potential = +500 mV)—filling the electron "vacancy". The electron transfer from photosystem II plastoquinone to the photosystem I reaction center occurs in several stages along an electrochemical gradient. At certain stages, electron transport in this region is coupled with proton translocation across the membrane and, consequently, ATP synthesis. The absorption of another light quantum by the chlorophyll molecule (P₇₀₀) in the photosystem I reaction center causes the ejection of an electron, which is delivered to an Fe-S protein molecule and subsequently transferred via a chain of carriers to NADP⁺.

Cyanobacteria (blue-green algae) are a large group of prokaryotes capable of oxygenic photosynthesis and endowed with diverse physiological capabilities. All of them possess Two Photosystems, use water as an exogenous electron donor, and evolve molecular oxygen. They contain chlorophyll a, phycobiliproteins, and carotenoids. At the same time, cyanobacteria have been shown to exhibit the capacity for anoxygenic photosynthesis, which is associated with the shutdown of photosystem II while photosystem activity I is maintained. Under these conditions, the exogenous electron donors are reduced sulfur compounds, molecular hydrogen, and A number of organic compounds (sugars, acids). The ability of cyanobacteria to switch from one type of photosynthesis to another highlights the remarkable flexibility of their light metabolism. Although most cyanobacteria are obligate phototrophs, an active endogenous metabolism has been detected in the dark, driven by glycogen accumulated during the light phase as an endogenous substrate. Glycogen is catabolized via the Pentose Phosphate Pathway or glycolysis, ensuring the Complete oxidation of a glucose molecule. In this process, hydrogen from NADH₂ is fed into the respiratory chain, with molecular oxygen serving as the terminal acceptor. It is also known that under anaerobic conditions, cyanobacteria are capable of sulfur respiration. The Krebs cycle in these bacteria is "incomplete" and serves a purely biosynthetic function. They assimilate CO₂ via the Calvin cycle, whereas organic compounds (acetate, pyruvate) are generally utilized only as supplementary carbon sources. Certain species are capable of chemoheterotrophic growth, albeit on a limited range of substrates (a few sugars). Over 250 cyanobacterial strains have been found to possess the ability to fix molecular nitrogen.

Prochlorophytes are prokaryotes capable of oxygenic photosynthesis. Unlike cyanobacteria, they lack phycobiliproteins. Their photosynthetic pigments are represented by chlorophylls a and b, along with carotenoids. The bulk of the latter consists of β-carotene and a xanthophyll structurally close to zeaxanthin. Other carotenoids are present in minor amounts: echinenone, β-cryptoxanthin, isocryptoxanthin, and others. All of these carotenoids are also found in cyanobacteria. Prochlorophytes fix carbon dioxide via the Calvin cycle. Representative species include Prochloron didemni (an exosymbiont of ascidians) and Prochlorothrix hollandica (free-living).