Dissimilatory sulfate reduction in the intestinal sulfate-reducing bacteria

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Dissimilatory sulfate reduction in the intestinal sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) are common in anaerobic areas of soils, wetlands, fresh and marine waters, and available in the microbiocenosis of large intestine of humans and animals [1,63]. These microorganisms, dissimilating sulfate to hydrogen sulfide, are involved in the process of biogeochemical sulfur cycle in nature [63]. The sulfate dissimilation process is called the „dissimilatory sulfate reduction” or „sulfate respiration” [54]. Intensive sulfate reduction by SRB, and accordingly the accumulation of toxic hydrogen sulfide in the intestine, is leading to the development of various diseases [12, 16, 46, 49, 55]. SRB and the products of their metabolism are often found during bloody diarrhea and abdominal pain [51, 55]. It is believed that they can cause weight loss, frequent defecation, arthritis, rheumatic diseases, increased intestinal permeability, ulcerative colitis, and malaise, in general [12, 13, 15, 17, 46, 49, 55].

Studying the process of dissimilatory sulfate reduction in the natural SRB strains and those that are isolated from the animal and human intestine during various diseases as well as comparing their biochemical, physiological, genetic and morphological properties is necessary for clarifying of the role of SRB in the development of various human diseases. It is also important to study the thermodynamic properties of electron donors, trophic relations in different species and genera of SRB, and their diversity in natural conditions. The research of SRB from intestines of humans and animals is conducted only in several leading laboratories in the world [12, 13, 48, 49, 50, 55]. The isolation and identification of new strains of intestinal SRB, the study of their physiological and biochemical characteristics, the development of the basic criteria for assessing the aggressiveness of the strains, the toxicity of products of their metabolism for the intestinal mucosa and the clarifying of their role in the disease development are currently most important.

The estimated number of SRB and the level of hydrogen sulfide accumulation in human feces can predict the progress of inflammation in the intestines. Analyzing the sulfate dissimilation process in the intestinal SRB strains allows a better understanding of their temporal dynamics at different stages of its reduction. The measuring of the aggressiveness of SRB and the intestinal mucosal toxicity of the products of their metabolism may be proposed as indicators. Experimental data allow developing basic criteria for the assessment of the course of the inflammatory process and establishing of the level of disease risk in order to prevent it. Such research is also promising for the development of methods of prevention against inflammatory bowel disease. The described physiological and biochemical parameters are important for creating animal models of inflammatory bowel disease involving SRB and using these models to study the mechanisms of the action of antimicrobial prophylactics and the therapy against specific components involved in the pathogenesis of the disease. New opportunities for studying of the inflammatory bowel disease and the assessment of the effectiveness of its treatment are extremely urgent problems in modern biology and medicine.

The aim of this review was to summarize the results of current research and generalize new data on the process of dissimilatory sulfate reduction in the intestinal sulfate- reducing bacteria based on own results and those from recent literature.

1. Intestinal sulfate-reducing bacteria and the bowel diseases. Sulfate-reducing bacteria Desulfotomaculum, Desulfobulbus, Desulfomicrobium, Desulfomonas, and Desulfovibrio genera belong to the intestinal microbiocenosis in humans and animals [1, 22, 23, 24, 46]. The knowledge on the interaction of SRB with other microorganisms in intestines is not sufficient. Bacteria living on the surface of the colon mucosa are in close relationship with the human body. They interact with the cells of the immune and neuroendocrine system more closely than microorganisms in the intestine lumen [12, 13, 19, 20]. It is believed that the species composition and the number of SRB on the surface of the intestinal mucosa differ from microorganisms in its lumen. The presence of sulfate ions promotes the growth of intestinal SRB which use molecular hydrogen and compete for this substrate with methanogenic bacteria [12, 13].

Among genera of SRB, the species of the Desulfovibrio genus in human and animal diseases are the most often isolated (Fig. 1). These bacteria are also isolated in the mono- and polymicrobial infections of the gastrointestinal tract [6, 20, 48, 51,52, 55, 63].

Bacteria Desulfovibrio genus isolated from different objects (light microscopy, x1,000): A - isolate caused a pyogenic liver abscess (photo by Tee et al. 1996) [71]; B - isolate caused bacteremia (photo by McDougall et al. 1996) [55]

intestine ulcerative colitis dissimilation

In 1976, W.E.C. Moore isolated SRB from human feces for the first time and identified the bacteria as Desulfomonas pigra which was subsequently reclassified to Desulfovibrio piger [1,4]. Similar research was carried out by J. Loubinoux et al. who isolated bacteria Desulfomonas and Desulfovibrio genera from the human intestine [48-51].

It is believed that SRB are not pathogenic in humans and animals [1]. However, they can cause various diseases together with other infections [12, 19, 20]. The most often isolated genus among SRB during the disease is the Desulfovibrio genus, including D. fairfieldensis. These bacteria may be pathogenic more than other species of SRB [51]. Bacteria D. fairfieldensis are isolated during mono- and polymicrobial infections of the gastrointestinal tract [51]. Loubinoux J. et al. found that 12 of 100 samples of purulent abdominal and pleural cavities contained human Desulfovibrio piger, D. fairfieldensis or D. desulfuricans [50]. Bacteria D. desulfuricans causing bacteremia was isolated from bleeding microvilli of the colon [51]. This research shows that the main way SRB penetrate the blood vessels is through damaged intestinal microvilli and then bacteria cause an infection. SRB is also detected in oral cavity [1,20]. Similarly to some metha- nogens, they can cause the development of other diseases, including cholecystitis, abscesses of the brain and abdomen, ulcerative enterocolitis, cancer, etc. [1,20, 46, 49].

Bacteria of Desulfotomaculum, Desulfobulbus, Desulfomicrobium, Desulfomonas, and Desulfovibrio genera in the anaerobic respiration, in addition to sulfate, can consume other electron acceptors, including elemental sulfur, fumarate, nitrate, dimethyl sulfoxide, Mn (IV) and Fe (III) [1, 19, 63]. Bacteria Desulfovibrio gigas are even capable of aerobic respiration [1]. However, aerobic conditions inhibit the process of dissimila- tory sulfate reduction in most SRB genera [47]. Therefore, SRB grow using sulfate reduction only in the environment with the absence of molecular oxygen [19]. They are strictly (obligate) anaerobic microorganisms present in anoxic environments that are rich in sulfates [1, 19, 63]. Such conditions are characteristic for wetlands, silt ponds and intestines of humans and animals [19, 63]. Thus, the high concentration of sulfate in marine and fresh waters as well as in human and animal intestine is creating favorable conditions for the SRB growth [5]. Under these conditions, sulfide formed in the SRB, is oxidized to sulfate by the chemolithotrophic or photolithotrophic bacteria, which are providing constant level of sulfate in the natural environments [63].

According to the nutritional requirements and the carbon and energy source, SRB may be divided into groups of chemoorganoheterotrophs, chemolithoheterotrophs and chemolithoautotrophs (Fig. 2) [1]. In the chemolithoheterotrophic or chemolithoautotro- phic conditions, the bacterial nutrition is provided by the oxidation of mainly hydrogen [90].

The chemolithoheterotrophic SRB include some species of the Desulfovibrio and Desulfotomaculum genera [4]. Bacteria of the Desulfovibrio genus grow due to the oxidation of molecular hydrogen using acetate and CO₂ to build carbon containing metabolites [19]. Bacteria D. vulgaris use acetate and CO₂ in the interrupted Krebs cycle with formation of acetyl which is transformed to pyruvate. Pyruvate in the presence of CO₂ is then transformed to oxaloacetate [1].

Chemolithoautotrophic type of nutrition is described in some species of the Desulfotomaculum, Desulfobacter, Desulfococcus and Desulfonema genera, and Archaeo- globus genus [1, 19].

Sulfate-reducing bacteria can use compounds such as lactate, pyruvate, formate, acetate, propionate, butyrate, fatty acids, ethanol, fructose, acetone, dicarboxylic acids and amino acids as a source of carbon and energy [19, 20, 63]. This way of getting nutrition is called chemoorganoheterotrophy. Besides these compounds, SRB can sometimes use carbon (IV) oxide which may be the only source of carbon for autotrophic growth. The dominant among SRB in human feces is the genus Desulfovibrio (D. fair- fieldensis, D. desulfuricans) [1, 19]. In some cases and with some frequency, bacteria Desulfobacter, Desulfotomaculum, and Desulfobulbus were also isolated. However, the species of Desulfotomaculum genus were seldom isolated and in small quantities compared to other SRB [19]. Prevalence of SRB varies in different people. These microorganisms were found in the feces of 70% of healthy people in the United Kingdom and only in 15% of the inhabitants of Africa. SRB number observed in the stool of 143 healthy people ranged from 10² to 10¹¹ cells/g of feces [1].

Another study with 87 healthy people found that the number of SRB ranged from 10⁷ to 10¹¹ cells/g of feces, and it differs among residents of different areas [12, 13]. As already mentioned, the species of the Desulfovibrio genus is dominant among SRB in the gut. They account for 67-91% of total SRB number. Significantly fewer bacteria are found from Desulfobacter (9-16%), Desulfobulbus (5-8%) and Desulfotomaculum (2%) genera [19]. SRB producing the largest number of hydrogen sulfide were isolated from feces of human distal colon. It is probably due to the reaction of the environment because the proximal part of the colon is acidic (pH<5.5) and the distal part is neutral [12, 13].

It has been found that SRB are available not only in feces but they also colonize the intestinal wall [1]. As a result, the samples from men and women acquired by the rectal biopsy contain from 10⁶ to 10⁷ CFU/g of bioptate. In the mucosa of some people, the number of Desulfovibrio bacterial genus changed by several orders during the period of 12 months [19]. It probably depended on the nutrition of these individuals. SRB colonize the intestines of humans right from the beginning of their lives [1]. The presence of bacteria of Desulfovibrio genus was detected in the feces of infants under the age of six months. The number of Desulfovibrio bacteria in these children which had been breastfed or bottle-fed was 3.7x10³ and 4.5x10⁴ cells/g of feces, respectively [12, 13, 19].

Intestinal microflora plays an important role in physiology of humans and animals and their metabolism. Microorganisms are directly involved in the process of food digestion including the metabolization of short chain fatty acids (SCFA). Intestinal bacteria have effect on the human physiological functions and health [1]. For example, the colonization in the gut provides resistance to pathogens and activation or neutralization of mutagenic compounds such as hydrogen sulfide [20].

In spite of the above, the definitive role of SRB in the development of intestinal diseases has not been well characterized and studied yet. That is why it is important to isolate new strains of intestinal SRB, investigate their substrates and the process of sulfate dissimilation in detail, and consequently, the accumulation of hydrogen sulfide as well as the role of these microorganisms in the development of diseases.

2. Substrates of sulfate-reducing bacteria in animals and humans intestine.

The cells of the intestinal mucosa, mucin and other secretions are permanently destroyed and can be used by the intestinal bacteria as a source of energy. However, human nutrition has a significant effect on the species composition of these microorganisms and their metabolic activity [1,20]. The main sources of carbon and energy for bacteria of the intestine are polysaccharides, namely starch and cellulose. They also use a significant amount of oligosaccharides and proteins. The main products of metabolism in the colon are acetate, short chain fatty acids (SCFA), propionate, butyrate, H₂ and CO₂. Among other products of fermentation, lactate, succinate, ethanol, and CH₄ are found in some people. Branched SCFA, amines, phenols, indoles, H₂S and thiols formed during the fermentation are present in the human gut [1]. Most of these products of fermentation are further metabolized by the intestinal microorganisms (Escherichia, Bifidobacterium, Lactobacillus, and Enterococcus) [19]. The study of SRB isolated from feces of people showed that these organisms are able to use a variety of substrates as electron donors with lactate, pyruvate, acetate and ethanol being the most often used [1].

Sulfates are poorly absorbed in the human intestine. In total, 2-9 mmol of sulfate from food reaches the colon daily. Most of them are reduced in the intestine because sulfates are usually detected in fecal secretions in a quantity of less than 0.5 mmol per day [19]. A large number of sulfate may be in the water and vegetables. Moreover, sulfur dioxide, sulfite, bisulfite, metabisulfite and sulfate are used as food additives. In many food products (beer, cheese, wine, bread, canned meat and vegetables, pickled products), sulfur dioxide (SO₂) can be detected where it serves as a conservator, antioxidant or whitening agent. Studies in vitro have shown that intestinal bacteria can also get sulfates from depolymerization and desulfurization of glycoproteins which have a high content of sulfates [12, 13].

Another sulfate containing molecule is chondroitin sulfate, an acidic mucopolysaccharide. It is distributed in the tissues of mammals and considered to be an important source of carbon and energy in the colon. This polymer also stimulates the growth of SRB and the accumulation of sulfide in fecal material [12].

Chondroitin sulfate and mucin are not directly absorbed by SRB. This process depends on saccharolytic activity of some intestinal microorganisms, for example Bifidobacterium, Lactobacillus, and Enterococcus. The most of the SRB are in microparticles of intestine containing goblet cells [1].

Lactate is a product of fermentation in the gut by the Bifidobacterium and Lactobacillus genera. In healthy people, the concentration of this metabolite is not more than a few mmol/kg of feces. The research of digestive mass content which was obtained directly from the intestine during the autopsy showed that lactate is synthesized mainly in the proximal part of intestine [12, 13]. Lactate is well absorbed in the colon and intestinal bacteria can metabolize this compound and keep its concentration low. The formation of lactate in the gut is mainly caused by fermentation of carbohydrates such as starch. A small quantity is formed by etching other polysaccharides. Lactate is an electron donor for SRB in the human intestine. Other microorganisms of the intestine use it much less compared to SRB [1]. A positive correlation between the concentrations of lactate and starch in the human intestine was observed. It is believed that food containing starch can be used by intestinal SRB in the presence of sufficient concentration of sulfates [19].

Hydrogen is one of the products of fermentation in the colon. Intestinal bacteria use protons for splitting sugars, amino acids and carbohydrates [13, 66]. According to theoretical calculations, the daily production of H₂ in human colon is more than 1 liter in the presence of 40-50 grams of carbohydrates. This parameter depends on the food consumed by humans. The total volume of gas in healthy people does not exceed this value. In total, 2.5-14% of H₂ is formed in the fermentation process. This discrepancy between theoretical and practical H₂ level allocation is the result of activities of many microbial communities using H₂ in the gut [1].

In the United Kingdom, SRB were either not found or their number was very little in about 30% of people who had a high intensity methanogenesis in the large intestine [13]. Hydrogen is the only electron donor for intestinal methanogenic bacteria Methano- brevibacter smithii. Therefore, there is a competition for molecular hydrogen between the SRB and methanogenic organisms. If sulfates are present in sufficient quantities, SRB inhibit the use of hydrogen by the methanogens in the dissimilatory sulfate reduction process [13, 19].

The amount of sulfate in the diet can have an effect on competition for the substrate (molecular hydrogen, lactate) between SRB and methanogenic organisms in the colon. In people with elevated levels of methane, the inclusion of 15 mmol of sulfate per day in the diet causes a reduced intensity of methanogenesis. Under these conditions, the number of methanogenic bacteria decreases by three orders, while the number of SRB in feces increases by three orders of magnitude. In the absence of sulfate in the diet, SRB was not found. Thus, the intensity of methanogenesis can be regulated by the introduction of sulfate, even if SRB are in the low concentration in the intestine [13].

Ability of SRB to use the H₂ as the electron donor can have a significant effect on fermentation in the colon. Sulfate at a concentration of 15 mM stimulates the growth of SRB in the gut. It also stimulates acetate and propionate fermentation, and inhibits butyrate fermentation. Under these conditions, lactate does not accumulate [12].

The dominant species among SRB in the intestine is Desulfovibrio desulfuricans which belongs to human colon microbiocenosis [12, 19, 21]. Analysis of biofilm from human bioptate showed that this species was mixed with many types of bacteria. After injecting the Desulfovibrio genus in biofilm, the changes of biofilm's metabolism were observed, including the formation of carbon dioxide, a significantly decreased total content of SCFA and acetate accumulation which are typical for the SRB activity. Under these conditions, lactate was not accumulated in the medium because it was used by the Desulfovibrio bacteria as an electron donor. In addition to increasing concentrations of acetate, butyrate content was reduced threefold. The syntrophic interactions were observed between D. desulfuricans and saccharolytic bacteria (Lactobacillus, Bifidobacterium, Enterococcus) localized in the colon. The reasons for the formation of such biofilms together with SRB are unclear. They form also on digestion remains in the intestinal lumen and mucosal surface [12, 19].

Thus, the most common substrates for SRB in human colon are lactate, pyruvate, acetate and ethanol which can be electron donors in the process of dissimilatory sulfate reduction. The presence of sulfate in the human diet suppresses methanogenesis and, accordingly, the number of methanogenic bacteria, and increases the amount of sulfate- reducing bacteria in the gut. To clarify the role of sulfate-reducing bacteria and their participation in various human and animal diseases, the process of dissimilatory sulfate reduction is necessary to be studied in the natural strains of SRB and the species of SRB isolated from human and animal colon during diseases and from healthy subjects. It is also important to compare their biochemical, physiological, genetic and morphological properties, and to investigate the possibility of using electron donors and their thermodynamic properties in the process of sulfate dissimilation by SRB, in general.

3. Electron donors of intestinal sulfate-reducing bacteria. From a wide range of many electrons donors, which SRB use in the process of dissimilatory sulfate reduction, formate is probably the only also oxidized in periplasm [1, 69]. Biochemical and genetic studies have shown that formate dehydrogenases from bacteria D. vulgaris are localized in the periplasm. They use polyheme cytochrome c as an electron acceptor. Oxidation of all other electron donors occurs in the cytoplasm or on the inside of the cytoplasmic membrane [19]. In the large intestine, the most common electron donors for SRB are lactate, acetate and propionate. They are formed by fermentation of substrates which humans consume [12]. The oxidation of electron donor can be divided for three process.

Lactate oxidation. Bacteria D. vulgaris grow using sulfate and lactate as an energy source. Lactate is not fully oxidized to acetate and the formation of intermediate compounds occurs: pyruvate, acetyl-CoA and acetyl phosphate [1,40].

2Lactate^- + SOf" + H+ = 2Acetate^- + CO₂ + HS^- + 2H₂O (1)

AG°' = -196.4 kJ/mol

This reaction probably consists of the following stages of reduction:

Lactate^- + 2cyt c₃(ox) + AmH+ = Pyruvate^- + 2cyt c₃(red) (2)

the catalysis of this reaction is carried out by membrane specific complex of lactate dehydrogenase with active centers localized in the cytoplasm [19];

Pyruvate^- + CoA-SH + Fd_ox = Acetyl-S-CoA + CO₂ + Fd_red^2- + 2H+ (3)

the catalysis of pyruvate in the cytoplasm is carried out by pyruvate: ferredoxin oxido- reductase, EC 1.2.7.1 [44];

Fdre_d^2- + 2H+ = Fdo_X + H2 + AmH+ (4)

this reaction is catalyzed by one of the two membrane specific complexes of hydroge- nases, EchABCDEF or CooMKLXUHF, which are ferredoxin specific and present in the cytoplasm [1, 56]. Redox potential (E⁰ = -500 mV) of the reaction of acetyl-CoA with CO₂/pyruvate is significantly lower than that of H+/H₂. Hydrogen is formed (reaction 4), diffuses in periplasm and reacts with cytochrome c₃;

H₂ + 2cyt c₃(ox) = 2cyt c₃(red)^-1 + 2H+ (5)

electrons formed by oxidation of lactate (E⁰ = -190 mV) are transferred through cytoplasmic membrane with the formation of AmH+ to periplasmic cytochrome c₃ (reaction 5). Reaction 5 is catalyzed by one of four periplasmic cytochrome specific hydro- genases [19]:

4cyt c₃(red)^-1 + 0.5 SOf = 4cyt c₃(ox) + 0.5 H₂S (6)

Cytochrome c₃ is reduced in cytoplasm, transferred to periplasm via transmembrane electron transfer and then oxidized again (reaction 6).

The transfer of electrons from lactate to cytochrome c₃ causes the formation of AmH+ (reactions 2 and 4). H₂ formation in the cytoplasm and re-oxidation in periplasm are together called the intraspecific transfer of hydrogen or the hydrogen cycle [1,57].

The sequence of reactions occurs in the presence of specific enzymes and electron carriers. Formed hydrogen is used again for growth of D. vulgaris in the medium with lactate and sulfate [64, 69]. In the absence of sulfates, formed H₂ is not used again.

The formation of H₂ from lactate is energy-dependent. The scheme of lactate oxidation to CO₂ is presented in Fig. 4. This process is inhibited by protonophores and arsenates. However, H₂ formation from pyruvate, which is oxidized to acetate and CO₂, is not inhibited by protonophores and arsenates and it does not require energy [19].

Intraspecific transfer of H₂ is probably also involved in the dissimilatory sulfate reduction in the presence of CO. Cytoplasmic carbon monoxide dehydrogenase which catalyzes ferre- doxin reduction in the presence of CO is described in some SRB [63].

In addition to intraspecies hydrogen transfer, formate is also capable of the electron transport from the cytoplasm to periplasm. D. vulgaris genome contains genes which are responsible for the synthesis of formate C-acetyltransferase (pyruvate formate-lyase, EC 2.3.1.54) that catalyzes the formation of acetyl-S-CoA and formate from pyruvate and CoA-SH [19].

Pyruvate formate-lyase is a cytoplasmic enzyme. Bacteria D. vulgaris have another enzyme, formate dehydrogenase, EC 1.2.2.1, localized in periplasm [64]. Formate formed from pyruvate penetrates through the cytoplasmic membrane due to proton symport. Before that, formate can be used as the electron donor in the dissimilatory sulfate reduction or the reduction of protons to H₂ [66]. This process is called the intraspecies formate transfer [1].

Oxidation of acetate (acetyl-CoA) to CO₂.

Some thermophilic sulfate-reducing archaea oxidize organic compounds, such as acetate, lactate, fatty acids, alkanes, benzoic acid etc., completely to CO₂ using sulfate as an electron acceptor [19]. Acetyl-S-CoA is an intermediate compound which forms from CO₂. Some SRB, including Desulfobacter postgatei, use citric acid cycle (CAC) for oxidation of acetyl-S-CoA to CO₂ [1]. Therefore, they can use intermediates of CAC, namely citrate, aconitate, isocitrate, 2-oxoglutarate, succinyl-CoA, succinate, fumarate, malate and oxaloacetate [63]. However, most SRB, including Archaeoglobus fulgidus, use oxidizing acetyl-CoA synthase (decarbonylase)/ carbon monoxide dehydrogenase, tetrahydrofolate (H₄F) or tetrahydromethanopterin (H4MPT) as C1-carrier. In this way, acetyl-CoA is oxidized while using carbon (II) oxide via intermediates: methyl-H₄F (methyl-H₄MPT) ^ methylene-H₄F (methylene-H₄MPT) ^ methenyl-H₄F (methenyl-H₄MPT) ^ N¹⁰-formyl-H₄F (N⁵-formyl-H₄MPT) ^ formate (for- myl-methanofuran) (Fig. 5) [57].

Oxidizing acetyl-S-CoA synthase (decarbonylase), EC 2.3.1.169/carbon monoxide dehydrogenase, EC 1.2.99.2 plays an important role in the process of dissimilatory sulfate reduction. Oxidation of organic compounds is accompanied by the redox potential which is more negative than those of adenosine-5'-phosphosulfate (APS)^C03 (-60 mV) and HCO3/HS^- (-116 mV). Citric acid cycle is only one way of oxidation of succinate to fumarate with the formation of redox potential +33 mV. Oxidation of succinate to fumarate with sulfate as the final electron acceptor needs energy directing reverse electron transport [1, 19].

SRB involve phosphotransacetylase and acetate kinase in the oxidation of acetate to CO₂. Phosphotransacetylase, EC 2.3.1.8 catalyzes the phosphorylation of acetyl- CoA after which acetyl phosphate is formed and then changed into acetate by acetate kinase, EC 2.7.2.1 [43, 45].

Oxidation of propionate. Propionyl-S-CoA, which is formed from propionate or during the oxidation of fatty acids, is oxidized through intermediates: methylmalonyl-S-CoA, succinyl-S-CoA, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-S-CoA (Fig. 6) [19].

Bacteria Desulfobulbus propionicus grow using propionate and sulfur [1]. Obviously, oxidation of methylmalonyl-S-CoA is more energetically favorable than the oxidation of propionyl-S-CoA through acrylic-S-CoA. Since the redox potential of acrylic-S- CoA/propionyl-S-CoA is +69 mV, it is more disadvantageous than that of fumarate/succinate (+33 mV) [19].

Thus, lactate, acetate and propionate are important electron donors for the SRB growth in human and animal intestines. Perhaps, the presence of electron donors and sulfate in the intestine can cause intense development of SRB which in turn will probably heighten the risk of ulcerative colitis due to the formation of hydrogen sulfide. Thermodynamic characteristics of the donor electrons are important for the study of the vital activity of SRB and the process of dissimilatory sulfate reduction.

Thermodynamic properties of electron donors. Reactions occurring with a change in the oxidation state of atoms or reactions between the oxidizing and reducing agent are called redox reactions. The power of oxidant and reductant is determined by the redox potential (E⁰). It depends on the changes in the concentrations of ions of H+ and OH^- in the

Redox potential of sulfate/HS^- in the presence of 1 M sulfate ions, 1 M HS^-, pH 7.0 and temperature +25 °C is -217 mV. It is slightly larger (-200 mV) for the concentration of SOf < 30 mM and HS^- < 1 mM. Organic compounds of plant and animal origin (carbo hydrates, fatty acids, alkanes, aromatic hydrocarbons) can be oxidized completely to CO₂ by some SRB (Desulfobacter, Desulfococcus, Desulfosarcina, Desulfonema genera). Other SRB (Desulfotomaculum and Desulfovibrio genera), which have only some enzymes of the Krebs cycle, oxidize these organic compounds only partially into acetate. Redox potential is changing in the process of oxidation of organic compounds which can be electron donors for SRB. Each of these potential donors of electrons is much more negative than -200 mV of the pair sulfate / HS^- [19].

In the natural environment, E⁰ (SOf'/HS^-) is greater than ДЕ' of the reducing agent and they are used together. However, there are exceptions, for example H+/H₂ and S⁰/ HS^-. Redox potential of H+/H₂ at pH 7.0 (concentration of H+ is 10^-7 M and constant) increases from -414 mV (partial pressure of H₂ is 10⁵ Pa) to -270...-300 mV (partial pressure of H₂ from 1 to 10 Pa). Therefore, the oxidation of acetate to CO₂ (E⁰ = -290 mV) with H⁺ as an electron acceptor (E⁰ = -270 mV at H₂ 1 Pa) is thermodynamically possible. Microorganisms are able to grow in this range of the redox potential [1].

Redox potential of S⁰/HS^- increases from -270 to -120 mV under different conditions. In this case, SRB can be found in sulfur containing environments where they can grow through the S⁰ reduction [19, 64].

Growth of SRB via dissimilatory sulfate reduction is accompanied by oxidation of the substrate together with the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate [36, 39].

In the process of substrate phosphorylation of organic compounds, „energy rich” intermediates are formed [1]. Transport of electrons causes the formation of transmembrane electrochemical proton gradient or gradient of sodium ions. It leads to the phosphorylation of ADP by membrane-bound ATP synthase [19, 36].

For many years, it has been believed that SRB can grow only in the presence of organic substrates which serve as electron donors for the dissimilatory sulfate reduction. However, in 1978 a discovery was made that Desulfovibrio vulgaris can grow in the presence of H₂ and sulfate as a single source of energy [2]. It is also believed that energy is produced in these organisms largely as a result of the substrate phosphorylation [1,63].

Substrate phosphorylation is possible only by oxidation of organic substrates. One exception from this rule is the oxidation of bisulfite to sulfate through energy-rich intermediate products including adenosine-5'-phosphosulfate (APS) [41]. Using this reaction, some SRB grow in the presence of bisulfite and oxidize it to sulfate which is then reduced to hydrogen sulfide [1,34].

Thus, sulfate-reducing bacteria grow using organic compounds that serve as a source of carbon and energy as well as the electron donors. Sulfate is the primary final electron acceptor. Oxidation of organic compounds causes a change of redox potentials. SRB can grow in the presence of H₂ and sulfate as the sole energy source. Substrate phosphorylation is possible only by oxidation of organic substrates.

4. Sulfate dissimilation and accumulation of hydrogen sulfide. The dissimilatory sulfate reduction is a complex and multistage process providing SRB cells with energy in the form of ATP. As mentioned before, they consume sulfate as a terminal electron acceptor and obtain energy for their growth due to the oxidation of organic compounds and hydrogen [1, 19, 36]. The final product of sulfate reduction is hydrogen sulfide [33].

The reduction of sulfate to hydrogen sulfide occurs through many intermediates and is an eight-electron process [63]. However, these intermediates are not released by SRB into the environment [19].

The enzymes of SRB involved in the process of dissimilatory sulfate reduction are localized in the cytoplasm and periplasm. At the beginning stages of sulfate reduction, absorption of sulfate occurs in bacterial cells [36]. While sulfate can be transported into the cells simultaneously with protons, some halophilic species of SRB can absorb sulfate together with the flow of sodium ions [1].

Dissimilatory sulfate reduction can be divided into six stages (Fig. 7).

Sulfate activation. Before sulfate is reduced, it is transported into bacterial cells and activated by reaction catalyzed by the enzyme ATP sulfurylase, EC 2.7.7.4 which transfers sulfate to the adenine monophosphate moiety of ATP to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP) The reaction is also reversible, and therefore, ATP can be formed from APS and PP_i [39, 42, 68].

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ATP sulfurylase can be found in the cells of many different organisms and it differs by its molecular weight and mono-, di-, tetra- or hexameric structure. Most ATP sulfury- lases consist of identical subunits containing cobalt and zinc ions (Fig. 8, A) [19]. However, in the bacteria E. coli, this enzyme has different subunits. ATP sulfurylases of D. desulfuricans and D. gigas are homotrimers with molecular weights of 141 and 147 kDa, respectively.

Bacteria of the Desulfovibrio genus contain cytoplasmic pyrophosphatase, EC 3.6.1.1 which catalyzes the cleavage of pyrophosphate to two phosphate ions (Fig. 8, B) [39]. In the process of pyrophosphate hydrolysis, energy is released in the form of a transmembrane proton potential [1].

Cytoplasmic reduction of adenosine-5'-phosphosulfate (APS). Sulfate activation leads to an increase of the redox potential from -516 mV to -60 mV [1]. Increase of E° provides the reduction of APS which serves as an electron acceptor. Bacteria of the Desulfovibrio genus contain cytoplasmic APS reductase (adenylyl-sulfate reductase, EC 1.8.99.2) that promotes the reduction of APS to sulfite or bisulfite and AMP [9, 11, 41]. APS reductase is also present in the cells of some purple and green bacteria and the Thiobacillus genus [19].

The structure of Zn-containing ATP sulfurylase of D. desulfuricans (A) [74] and the structure of pyrophosphatase (B) [75]

Структура Zn-вмісної АТФ сульфу- рилази D. desulfuricans (A) [74] і структура пірофосфатази (B) [75]

APS reductase is a nonheme iron-sulfur containing flavoprotein with a molecular weight of 95 kDa which consists of a- and p-subunits (Fig. 9). The first (a) subunit contains a molecule of flavin adenine dinucleotide (FAD) and the second (P) contains two [4Fe-4S]-centers [1,9, 11]. This enzyme reduces APS to sulfite in the position N(5)-FAD. The substantially increased number of polar interactions between the protein matrix and cluster B compared to cluster C can explain the differences in the redox potential [59].

APS reductase can be isolated from the cells of D. desulfuricans and D. vulgaris, and found in phototrophic and denitrifying bacteria. In the denitrifying bacteria, enzyme converts sulfite and AMP to APS in the process of photosynthesis or denitrification [63]. The activity of this enzyme of bacteria D. vulgaris depends on various chemical and physical factors, particularly on adding salts in concentrations of 0.5-1.0 M which leads to its inactivation. Kinetics of direct and reverse reaction also depends on the concentration of the enzyme [41]. The reverse reaction is described by Michaelis-Menten kinetics. Increase of AMP concentration in the environment leads to the inhibition of the reverse reaction and the concentration of 1.8 mM AMP and more causes the reaction to be terminated [1, 19].

The dissimilatory sulfate reduction to H₂S in bacteria D. vulgaris occurs through the formation of sulfite as an intermediate product [64].

Cytoplasmic reduction of sulfite. The next important stage in the process of dissimilatory sulfate reduction is sulfite which is the product of the reduction of APS [19]. Sulfite ( ) is more reactive than sulfate. Reduction of SO3" to S^2- is carried out by the enzyme called dissimilatory sulfite reductase, EC 1.8.99.1 (Fig. 10) [34, 59].

This enzyme is usually composed of two a- and p-subunits (a2p2). However, the bacteria D. vulgaris and D. desulfuricans Essex contain a third subunit (y). It has been proven that dissimilatory sulfite reductase in these microorganisms is a hexamer (a2p2y2) [1].

Active centers of sulfite reductases have two metal ion cofactors, siroheme and [FeS]-cluster [7, 19] (Fig. 11). They are involved in the transport of electrons. Six electrons are transported in the process of the reduction of sulfite to sulfide [8, 10, 63].

SRB have the following main types of dissimilatory sulfite reductases: desulfoviri- dine, desulforubidine, desulfofuscidine, and protein P₅₈₂ [19].

Bisulfite is one form of sulfite. Some scientists believe that the actual substrate in the process of dissimilatory sulfite reduction to sulfide is bisulfite rather than sulfite [1]. That is why sulfite reductase is often also called bisulfite reductase [63].

For many years, there has been a controversy around the following equation:

HCO3 + № + 6H+ = HS^- + 3H₂O; E⁰ = -116 mV (9)

because bisulfite reductase also catalyzes reactions 9 and 10 in the high concentration of HCOi [1].

3HSO₃^- + 2e + 3H+ = S₃CЈ + 3H₂O; E⁰ = -173 mV (10)

S₃C% + 2e + H+ = S₂CЈ + ; E⁰ = +225 mV (11)

According to one of the hypotheses, SRB contain thiosulfite reductase which catalyzes the reaction [19]:

+ 2b + H+ = HS^- + НС O3; E⁰ = -402 mV (12)

Two hypotheses regarding sulfite reduction have been suggested [1,64]:

• Consistent reduction through three two-electron steps with the formation of trithi- onate and thiosulfate as intermediate compounds;

• Direct six-electron reduction without the formation of trithionate and thiosulfate as intermediates.

Sulfite reductase plays an important role in the process of assimilation of sulfur. The enzyme promotes the formation of sulfide for synthesis of sulfur-containing amino acids including methionine and cysteine. This enzyme found in the cells of Desulfovibrio genus as well as in many other SRB [19, 64].

The mechanism of the six-electron sulfite reduction involves Fe²+ which connects sulfur atom with sulfite ion [1]. The two-electron reduction causes the oxygen atom in SO-bond to be protonated and then hydroxyl anion can be eliminated [7, 10]. Sulfide is formed after repeated reduction by two electrons and the subsequent protonation of oxygen atoms which are then gradually removed from the atoms of sulfur.

D. vulgaris bacteria contain four periplasmic hydrogenases, including three [NiFe]- hydrogenases, EC 1.12.99.6 and one [FeFe]-hydrogenase, EC 1.12.7.2 [1, 58]. While the three [NiFe]-hydrogenases are bound with the main periplasmic polyheme cytochrome c-type (TpI-c3), one of them, [NiFe]-hydrogenase 2, is probably also bound with the second polyheme cytochrome c (TpII-c3). If bacteria grow in the medium which contains a small amount of nickel and in the presence of hydrogen and sulfate then only [FeFe]-hydrogenase is synthesized. Under these conditions, the level of biomass accumulation is practically unchanged [19]. It has been established that removing of gene of [FeFe]-hydrogenase or one of [NiFe]-hydrogenases also do not have an effect on the growth of bacteria D. vulgaris [14, 18]. These data show that the four hydrogenases can completely functionally change each other, especially when growing the bacteria in the medium with high concentrations of H₂ [62].

Transmembrane electron transfer. In the periplasm, protons are transported to the cytochrome c₃ by periplasmic hydrogenases [1]. Subsequently, electrons from cytochrome c₃ are transferred through the cytoplasmic membrane to the recovered APS and HSO3 in cytoplasm. Electron transport through membranes involves a protein hmc-com- plex [19]. This complex is on the one side associated with periplasmic region of polyheme cytochrome c, and on the other with the cytoplasmic side containing FeS-protein (Fig. 15). It has a structure similar to heterodisulfite reductase. The hmc-complex is the most studied in bacteria D. vulgaris [60]. After removing hmc genes, bacteria D. vulgaris grew only in the medium with lactate and sulfate, and the growth significantly slowed down in the presence of only H₂ and sulfate. The genome of D. vulgaris encodes transmembrane protein complexes (TpII-c3 and Hme) [1,62].

They are similar to the hmc-complex and located on periplasmic side of cytochrome c and cytoplasmic side with FeS-proteins [53]. The sequences of these proteins also resemble those of heterodisulfite reductase [19]. Three transmembrane complexes (Hmc, Hme and TpII-c3) can likely carry out similar functions to four periplasmic hydro- genases [64].

M.S. Sim et al. (2013) have tested mutant strains lacking one or two periplasmic (Hyd, Hyn-1, Hyn-2, and Hys) or cytoplasmic hydrogenases (Ech and CooL), and a mutant strain lacking type I tetraheme cytochrome (TpI-c₃). They have shown that wild- type D. vulgaris and its hydrogenase mutants had comparable growth kinetics and produced the same sulfur isotope effects. In continuous culture, wild-type D. vulgaris and the CycA mutant produced similar sulfur isotope effects, underscoring the influence of environmental conditions on the relative contribution of hydrogen cycling to the electron transport. The schematic representation of two proposed pathways for electron transport during sulfate reduction in D. vulgaris Hildenborough is below presented in Fig. 16 [67].

In D. vulgaris genome, a cluster of gene encoding transmembrane protein complex (Qmo complex) was found. There are no genes encoding periplasmic cytochrome c [1, 19]. The complex Qmo is involved in the reduction of APS (Fig. 17) [65]. Heterodisulfide reductase, EC 1.8.98.1 of methanogens catalyzes the reduction of heterodisulfide of CoM-SS-CoB to coenzyme M (HS-CoM) and coenzyme B (HS-CoB). Both coenzymes are missing in the SRB. Cell extracts of SRB do not catalyze the reaction of CoM-SS-CoB and oxidation of CoM-SH + CoB-SH [60, 61].

Ramos A.R. et al. (2012) have reported the first direct evidence that QmoABC and AprAB interact in Desulfovibrio spp., using co-immunoprecipitation, cross-linking Far- Western blot, tag-affinity purification, and surface plasmon resonance studies. They showed that the QmoABC-AprAB complex has a strong steady-state affinity, but has a transient character due to a fast dissociation rate. Far-Western blot identified QmoA as the Qmo subunit most involved in the interaction. Nevertheless, electron transfer from menaquinol analogs to APS through anaerobically purified QmoABC and AprAB could not be detected. Authors propose that this reaction requires the involvement of a third partner to allow electron flow driven by a reverse electron bifurcation process. This process is deemed essential to allow coupling of APS reduction to chemiosmotic energy conservation [65].

Ramos A.R. et al. (2012) have proposed a schematic representation of the QmoABC- AprAB interaction and the involvement of third partners. In the hypothesis of an electron bifurcation process, the putative electron acceptor of QmoB with a high redox potential is represented by a question mark (Fig. 17A). In the hypothesis of an electron confurcating, mechanism several possible co-electron donors for the Qmo complex are considered: ferredoxin (Fd), hydrogenase (Hase), formate dehydrogenase (Fdh) or NADH dehydrogenase (Nox) (Fig. 17B). The soluble HdrABC-MvhGAD complex (Fig. 17C) and the membrane-bound HdrED (Fig. 17D) of methanogens are shown for comparison. The gray dashed arrows represent electron bifurcation in (A, C), or electron confurcation in (B). The gray boxes represent the cytoplasmic membrane with + indicating the periplasm and - the cytoplasm (Fig. 17) [65].

FeS proteins are a group of proteins involved in the processes of electron transport (ferredoxins) and some enzymes that catalyze different redox reaction [19]. Depending on the structural features of FeS centers, ferredoxins can carry out simultaneous transfer of one or two electrons [7, 10]. Redox potential of ferredoxins is preferably in the range of -490 to -310 mV. However, there have been described some FeS proteins with positive redox potential of +350 mV [1].

Ferredoxins play an important role in the metabolism of SRB, combining catabolic processes together with biosynthetic reactions. Physiological reactions in SRB cells occur at the negative redox potentials. Under these conditions, FeS proteins are important for the functioning of enzymes, and used as carriers of electrons (Fig. 18) [19].

FeS proteins in SRB have an amino acid sequence similar to heterodisulfide reductase. Perhaps, they have different substrate specificity and can be involved in other functions. In methanogenic archaea, the reduction of H₂ occurs through oxidation of methyl-coenzyme M. The concentration of H₂ under these conditions decreases and methane is produced. It is believed that disulfide/HS^- couple in SRB can be also involved in the transfer of electrons from hydrogen to sulfite [1].

Cytoplasmic oxidation of molecular hydrogen. The process of molecular hydrogen oxidation occurs by involving cytoplasmic hydrogenase and FeS proteins [19]. Bacteria D. vulgaris contains two membrane complexes of hydrogenase, EchABCDEF and CooMKLXUHF, which are interrelated [14, 18, 56]. They catalyze the reduction of ferredoxins in the presence of H₂ or protons to H₂ through ferredoxin reduction. Both of these reductions cause the formation of proton electrochemical potential (AmH⁺) (energy controlled reverse electron transfer) [58].

Hydrogenase catalyzes the oxidation of H₂ and reduction of ferredoxin [66]. For the studying hydrogenases of SRB, it is necessary to consider the bacterial growth in H₂ and sulfate in the presence of acetate and CO₂ as carbon sources. They are used by the cells forming acetyl-phosphate, acetyl-S-CoA and pyruvate [19]. Acetyl-S-CoA is formed from pyruvate in the reduction reaction of carboxylation involving pyruvate:ferredoxin oxidore- ductase [44]. Redox potential (E⁰) of acetyl-S-CoA + CO₂/pyruvate is -500 mV and, therefore, considerably more negative than H+/H₂ pairs (-270 to -300 mV), especially if the partial pressure of H₂ is very low (1 to 10 Pa) [1]. For the synthesis of pyruvate from acetyl-CoA, CO₂ and H₂ are necessary so that electrons from H₂ may have a more negative potential. This is achieved by a reverse energy transfer of electrons from H₂ to fer- redoxin involving hydrogenases [60]. This pattern is characteristic for other reduction reactions such as reductive carboxylation of succinyl-S-CoA to 2 oxoglutarate (-500 mV) or reduction of CO₂ to CO (-520 mV) [1, 19].