Disulfide bond-dependent mechanism of protection against oxidative stress in pyruvate-ferredoxin oxidoreductase of anaerobic Desulfovibrio bacteria

Oxidative decarboxylation of pyruvate forming acetyl-coenzyme A is a crucial step in many metabolic pathways. In most anaerobes, this reaction is carried out by pyruvate-ferredoxin oxidoreductase (PFOR), an enzyme normally oxygen sensitive except in Desulfovibrio africanus (Da), where it shows an abnormally high oxygen stability. Using site-directed mutagenesis, we have specified a disulfide bond-dependent protective mechanism against oxidative conditions in Da PFOR. Our data demonstrated that the two cysteine residues forming the only disulfide bond in the as-isolated PFOR are crucial for the stability of the enzyme in oxidative conditions. A methionine residue located in the environment of the proximal [4Fe-4S] cluster was also found to be essential for this protective mechanism. In vivo analysis demonstrated unambiguously that PFOR in Da cells as well as two other Desulfovibrio species was efficiently protected against oxidative stress. Importantly, a less active but stable Da PFOR in oxidized cells rapidly reactivated when returned to anaerobic medium. Our work demonstrates the existence of an elegant disulfide bond-dependent reversible mechanism, found in the Desulfovibrio species to protect one of the key enzymes implicated in the central metabolism of these strict anaerobes. This new mechanism could be considered as an adaptation strategy used by sulfate-reducing bacteria to cope with temporary oxidative conditions and to maintain an active dormancy.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: A review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

How obligatory is anaerobiosis?

Historically many bacteria have been classified as obligate anaerobes. They have been construed as wholly intolerant of oxygen, a feature that was originally ascribed to their lack of superoxide dismutases and catalases. Clostridial species were regarded as classic examples. We now know that this view is quite wrong: enzymes that scavenge superoxide, hydrogen peroxide and even oxygen itself abound in anaerobes. In the current issue of Molecular Microbiology, Hillmann et al. demonstrate that full production of these proteins can allow Clostridium acetobutylicum to survive and even grow in oxygenated culture medium. Evidently, oxidative defences in anaerobes can be robust. In all likelihood, they are critical for the movement of bacteria through aerobic environments to new anaerobic habitats.     

The oxygen tolerance of sulfate-reducing bacteria isolated from North Sea waters

Five strains of sulfate-reducing bacteria, previously isolated from North Sea waters and identified asDesulfovibrio vulgaris, were investigated for their abilities to survive in aerobic natural seawater. Viable organisms of all strains were recoverable after exposure to oxygen for more than 72 h. The level of the protective enzymes superoxide dismutase and catalase detectable in these strains and the low rates at which oxygen was reduced probably account at least in part for their considerable abilities to survive in aerobic environments. The autoxidizable nature of cytochromec ₃ and KCN-inhibitable cytochromec oxidase activity present in these bacteria are postulated to act as possible oxygen-scavenging mechanisms analogous to the activity of NADH oxidase present in certain other strict anaerobes.     

Unusual reactions involved in anaerobic metabolism of phenolic compounds

Aerobic bacteria use molecular oxygen as a common co-substrate for key enzymes of aromatic metabolism. In contrast, in anaerobes all oxygen-dependent reactions are replaced by a set of alternative enzymatic processes. The anaerobic degradation of phenol to a non-aromatic product involves enzymatic processes that are uniquely found in the aromatic metabolism of anaerobic bacteria: (i) ATP-dependent phenol carboxylation to 4-hydroxybenzoate via a phenylphosphate intermediate (biological Kolbe-Schmitt carboxylation); (ii) reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA; and (iii) ATP-dependent reductive dearomatization of the key intermediate benzoyl-CoA in a 'Birch-like' reduction mechanism. This review summarizes the results of recent mechanistic studies of the enzymes involved in these three key reactions.     

转录调控因子Rex的功能及调节机制研究进展

转录因子Rex是一种广泛存在于革兰氏阳性菌,能够与NADH或者NAD⁺直接结合响应胞内NADH/NAD⁺的氧化还原传感器,与靶基因的结合可调节细胞内的多种生理代谢。NAD（H）是调节细胞能量代谢的必需辅酶,显示微生物细胞内的氧化还原状态。研究发现Rex的调节活性与细胞内NADH/NAD⁺比率相关。需氧和厌氧菌属中Rex单体和复合物晶体结构的解析揭示了Rex、NADH/NAD⁺和靶基因间的作用关系及调控机制。通过比较分析了不同菌株中Rex单体和复合物的晶体蛋白结构,并揭示了NADH/NAD⁺对Rex调控活性的影响,进一步解析了Rex与碳和能量代谢、厌氧代谢、发酵、生物膜等之间的联系,并展望了Rex的研究和应用方向。     

Oral anaerobes cannot survive oxygen stress without interacting with facuItative/aerobic species as a microbial commmunity

D.J. Bradshaw, P.D. Marsh, G.K. Watson and C. Allison. 1997. Anaerobic bacteria are found commonly as components of mixed culture biofilms in many aerated habitats, including the mouth. Previous studies showed that anaerobes could survive in planktonic and biofilm communities in aerated conditions when part of a community including facultative and/or aerobic species, and the numbers and proportions of anaerobic species increased as biofilms aged. When the obligate anaerobes were grown in the absence of aerobic/facultative species, however, they were unable to grow in either the planktonic or biofilm culture. The mean survival times of organisms in the aerated culture containing four anaerobic species varied from around 5 min for Fusobacterium nucleatum and Veillonella dispar , to less than 4 min for Porphyromonas gingivalis and Prevotella nigrescens . In addition, in this culture, the biofilm mode of growth did not provide a haven for these bacteria in the absence of oxygen-consuming species.     

Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.     

Oxidative stress response in the opportunistic oral pathogen Fusobacterium nucleatum

The anaerobic, Gram-negative bacillus Fusobacterium nucleatum plays a vital role in oral biofilm formation and the development of periodontal disease. The organism plays a central bridging role between early and late colonizers within dental plaque and plays a protective role against reactive oxygen species. Using a two-dimensional gel electrophoresis and mass spectrometry approach, we have annotated 78 proteins within the proteome of F. nucleatum subsp. nucleatum and identified those proteins whose apparent intracellular concentrations change in response to either O(2)- or H(2)O(2)-induced oxidative stress. Three major protein systems were altered in response to oxidative stress: (i) proteins of the alkyl hydroperoxide reductase/thioredoxin reductase system were increased in intracellular concentration; (ii) glycolytic enzymes were modified by oxidation (i.e. D-glyceraldehyde 3-phosphate dehydrogenase, and fructose 6-phosphate aldolase) or increased in intracellular concentration, with an accompanying decrease in ATP production; and (iii) the intracellular concentrations of molecular chaperone proteins and related proteins (i.e. ClpB, DnaK, HtpG, and HrcA) were increased.     

Catalase and Superoxide Dismutase: Distribution, Properties, and Physiological Role in Cells of Strict Anaerobes

This review considers the distribution of the main enzymes of antioxidative defense, superoxide dismutase (SOD) and catalase, in various groups of strictly anaerobic microorganisms: bacteria of the genus Clostridium, Bacteroides, sulfate-reducing and acetogenic bacteria, methanogenic archaea, etc. Molecular and biochemical properties of purified Fe-containing SODs, cambialistic SODs, and heme catalases are presented. The physiological role and origin of the enzymes of antioxidative defense in strict anaerobes are discussed. Physiological responses (induction of SOD and catalase) to factors provoking oxidative stress in the cells of strict anaerobes able to maintain viability under aerobic conditions are also considered.     

The nature of anaerobic fungi and their polysaccharide degrading enzymes

Conclusion The discovery of anaerobic fungi has added a new member to the indigenous microorganisms that inhabit the rumen ecosystem. Anaerobic fungi do not appear essential for the survival of ruminants due to their presence in very low numbers, and sometimes absence, in ruminants fed low fiber diets, but their presence may likely be very important in the digestion of fibrous diets. The anaerobic fungi have adapted well to the rumen environment. They are able to ferment a large array of soluble carbohydrates and can synthesize cellular components in an anaerobic environment. The fungi posses hydrogenosomes for the removal of reducing equivalents in the form of molecular hydrogen and the removal of trace oxygen is a accomplished via removal by NADH oxidase. Their positive synergistic interaction with methanogenic bacteria eludes to their highly evolved role in the rumen environment. The fungi also produce resistant sporangia that allows for transfer of species to a new host in an oxygen environment. The anaerobic fungi posses a highly active array of polysaccharide degrading enzymes that may provide an advantage in the highly competitive rumen ecosystem. The production of specific enzymes that hydrolyze the lignocellulosic fraction of plant walls is unique in rumen microorganisms and allows for their attachment and growth on fibrous plant particles that are not available to the rumen bacteria.     

Physiology and biochemistry of waterlogging tolerance in plants

Waterlogging is a serious problem, which affects crop growth and yield in low lying rainfed areas. The main cause of damage under waterlogging is oxygen deprivation, which affect nutrient and water uptake, so the plants show wilting even when surrounded by excess of water. Lack of oxygen shift the energy metabolism from aerobic mode to anaerobic mode. Plants adapted to waterlogged conditions, have mechanisms to cope with this stress such as aerenchyma formation, increased availability of soluble sugars, greater activity of glycolytic pathway and fermentation enzymes and involvement of antioxidant defence mechanism to cope with the post hypoxia/anoxia oxidative stress. Gaseous plant hormone ethylene plays an important role in modifying plant response to oxygen deficiency. It has been reported to induce genes of enzymes associated with aerenchyma formation, glycolysis and fermentation pathway. Besides, nonsymbiotic-haemoglobins and nitric oxide have also been suggested as an alternative to fermentation for maintaining lower redox potential (low NADH/NAD ratio), and thereby playing an important role in anaerobic stress tolerance and signaling.     

The 'strict' anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain

Sulfate-reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane-bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O(2) min(-1) mg protein(-1), respectively). This 'strict anaerobic' bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought.     

Fluoride and organic weak acids as modulators of microbial physiology

Fluoride is widely used as an anticaries agent in drinking water and a variety of other vehicles. This use has resulted in major health benefits. However, there are still open questions regarding the mechanisms of anticaries action and the importance of antimicrobial effects in caries reduction. Fluoride acts in multiple ways to affect the metabolism of cariogenic and other bacteria in the mouth. F(-)/HF can bind directly to many enzymes, for example, heme-containing enzymes or other metalloenzymes, to modulate metabolism. Fluoride is able also to form complexes with metals such as aluminum or beryllium, and the complexes, notably AlF(4)(-) and BeF(3)(-).H(2)O, can mimic phosphate with either positive or negative effects on a variety of enzymes and regulatory phosphatases. The fluoride action that appears to be most important for glycolytic inhibition at low pH in dental plaque bacteria derives from its weak-acid properties (pK(a)=3.15) and the capacity of HF to act as a transmembrane proton conductor. Since many of the actions of fluoride are related to its weak-acid character, it is reasonable to compare fluoride action to those of organic weak acids, including metabolic acids, food preservatives, non-steroidal anti-inflammatory agents and fatty acids, all of which act to de-energize the cell membrane by discharging DeltapH. Moreover, with the realization that the biofilm state is the common lifestyle for most microorganisms in nature, there is need to consider interactions of fluoride and organic weak acids with biofilm communities. Hopefully, this review will stimulate interest in the antimicrobial effects of fluoride or other weak acids and lead to more effective use of the agents for disease control and other applications.     

Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum

It has been proposed that in some anaerobic facultatively autotrophic bacteria the acetyl CoA/CO dehydrogenase pathway is operating both in the reductive and in the oxidative direction, depending on the growth conditions. One of these anaerobes, the Gram-negative sulfate-reducing cubacterium Desulfobacterium autotrophicum, was examined for enzymes of the proposed pathway. All the required enzyme activities were present in sufficient amounts both in autotrophically and in heterotrophically grown cells, provided that the cellular tetrahydropterin rather than tetrahydrofolate was used as cosubstrate in some of the enzyme assays. The question arises whether two sets of enzymes are operating in the reductive and oxidative direction, respectively. The key enzyme of this pathway, CO dehydrogenase, which was reasonably oxygen stable, was analysed by native polyacrylamide gel electrophoresis and anaerobic activity staining. Extracts from heterotrophically grown cells exhibited five enzyme activity bands. Extracts from autotrophically grown cells showed the same pattern but an additional activity band appeared.     

严格厌氧菌铁氧还蛋白的研究进展

铁氧还蛋白(ferredoxin,Fd)是一类含有铁硫簇的小分子蛋白质,广泛存在于自然界中,参与生物体内的呼吸、发酵、固氮、二氧化碳固定和制氢等生理过程.Fd对于严格厌氧的细菌尤为重要,是因为这类细菌的能量代谢高度依赖于低氧化还原电势的生物组分,而Fd能够利用铁硫中心灵活调节其氧还电势,适应低电势需求.本文选取厌氧细菌...