Selenium respiration in anaerobic bacteria: Does energy generation pay off?

Selenium (Se) respiration in bacteria was revealed for the first time at the end of 1980s. Although thermodynamically-favorable, energy-dense and documented in phylogenetically-diverse bacteria, this metabolic process appears to be accompanied by a number of challenges and numerous unanswered questions. Selenium oxyanions, SeO42- and SeO32-, are reduced to elemental Se (Se0) through anaerobic respiration, the end product being solid and displaying a considerable size (up to 500 nm) at the bacterial scale. Compared to other electron acceptors used in anaerobic respiration (e.g. N, S, Fe, Mn, and As), Se is one of the few elements whose end product is solid. Furthermore, unlike other known bacterial intracellular accumulations such as volutin (inorganic polyphosphate), S0, glycogen or magnetite, Se0 has not been shown to play a nutritional or ecological role for its host. In the context of anaerobic respiration of Se oxyanions, biogenic Se0 appears to be a by-product, a waste that needs proper handling, and this raises the question of the evolutionary implications of this process. Why would bacteria use a respiratory substrate that is useful, in the first place, and then highly detrimental? Interestingly, in certain artificial ecosystems (e.g. upflow bioreactors) Se0 might help bacterial cells to increase their density and buoyancy and thus avoid biomass wash-out, ensuring survival. This review article provides an in-depth analysis of selenium respiration (model selenium respiring bacteria, thermodynamics, respiratory enzymes, and genetic determinants), complemented by an extensive discussion about the evolutionary implications and the properties of biogenic Se0 using published and original/unpublished results.

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy.

Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.

Anaerobic Degradation of Benzene and Polycyclic Aromatic Hydrocarbons.

Aromatic hydrocarbons such as benzene and polycyclic aromatic hydrocarbons (PAHs) are very slowly degraded without molecular oxygen. Here, we review the recent advances in the elucidation of the first known degradation pathways of these environmental hazards. Anaerobic degradation of benzene and PAHs has been successfully documented in the environment by metabolite analysis, compound-specific isotope analysis and microcosm studies. Subsequently, also enrichments and pure cultures were obtained that anaerobically degrade benzene, naphthalene or methylnaphthalene, and even phenanthrene, the largest PAH currently known to be degradable under anoxic conditions. Although such cultures grow very slowly, with doubling times of around 2 weeks, and produce only very little biomass in batch cultures, successful proteogenomic, transcriptomic and biochemical studies revealed novel degradation pathways with exciting biochemical reactions such as for example the carboxylation of naphthalene or the ATP-independent reduction of naphthoyl-coenzyme A. The elucidation of the first anaerobic degradation pathways of naphthalene and methylnaphthalene at the genetic and biochemical level now opens the door to studying the anaerobic metabolism and ecology of anaerobic PAH degraders. This will contribute to assessing the fate of one of the most important contaminant classes in anoxic sediments and aquifers.

Modulation of small intestinal homeostasis along with its microflora during acclimatization at simulated hypobaric hypoxia.

At high altitude (HA) hypobaric hypoxic environment manifested several pathophysiological consequences of which gastrointestinal (GI) disorder are very common phenomena. To explore the most possible clue behind this disorder intestinal flora, the major player of the GI functions, were subjected following simulated hypobaric hypoxic treatment in model animal. For this, male albino rats were exposed to 55 kPa (approximately 4872.9 m) air pressure consecutively for 30 days for 8 h/day and its small intestinal microflora, their secreted digestive enzymes and stress induced marker protein were investigated of the luminal epithelia. It was observed that population density of total aerobes significantly decreased, but the quantity of total anaerobes and Escherichia coli increased significantly after 30 days of hypoxic stress. The population density of strict anaerobes like Bifidobacterium sp., Bacteroides sp. and Lactobacillus sp. and obligate anaerobes like Clostridium perfringens and Peptostreptococcus sp. were expanded along with their positive growth direction index (GDI). In relation to the huge multiplication of anaerobes the amount of gas formation as well as content of IgA and IgG increased in duration dependent manner. The activity of some luminal enzymes from microbial origin like a-amylase, gluco-amylase, proteinase, alkaline phosphatase and beta-glucuronidase were also elevated in hypoxic condition. Besides, hypoxia induced in formation of malondialdehyde along with significant attenuation of catalase, glutathione peroxidase, superoxide dismutase activity and lowered GSH/GSSG pool in the intestinal epithelia. Histological study revealed disruption of intestinal epithelial barrier with higher infiltration of lymphocytes in lamina propia and atrophic structure. It can be concluded that hypoxia at HA modified GI microbial imprint and subsequently causes epithelial barrier dysfunction which may relate to the small intestinal dysfunction at HA.

Carbon monoxide. Toxic gas and fuel for anaerobes and aerobes: carbon monoxide dehydrogenases.

Carbon monoxide (CO) pollutes the atmosphere and is toxic for respiring organisms including man. But CO is also an energy and carbon source for phylogenetically diverse microbes living under aerobic and anaerobic conditions. Use of CO as metabolic fuel for microbes relies on enzymes like carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which catalyze conversions resembling processes that eventually initiated the dawn of life.CODHs catalyze the (reversible) oxidation of CO with water to CO2 and come in two different flavors with unprecedented active site architectures. Aerobic bacteria employ a Cu- and Mo-containing CODH in which Cu activates CO and Mo activates water and takes up the two electrons generated in the reaction. Anaerobic bacteria and archaea use a Ni- and Fe-containing CODH, where Ni activates CO and Fe provides the nucleophilic water. Ni- and Fe-containing CODHs are frequently associated with ACS, where the CODH component reduces CO2 to CO and ACS condenses CO with a methyl group and CoA to acetyl-CoA.Our current state of knowledge on how the three enzymes catalyze these reactions will be summarized and the different strategies of CODHs to achieve the same task within different active site architectures compared.

Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla.

BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like hybrid type II/III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO(2) fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.

Early warning signs of bulking in an activated sludge system through interpretation of ATP data in a systems analysis context.

A research project was undertaken at an integrated thermomechanical pulp and paper mill in Canada to evaluate the use of adenosine triphosphate (ATP) monitoring methods in order to identify the potential for operational problems related to microbiological aspects of activated sludge. The specific filamentous bulking ATP (fbATP) ratio is an emerging measurement technique that measures the proportion of flocs that have bulking potential by filtering a sample through a 250 microm mesh and measuring the ATP in the retentate. For the host mill in this study, the specific fbATP measurement provides early warning signs of bulking, at 1.0 to 1.5 times the sludge age, before poor settling occurs. A possible bulking scenario was identified in which the initiator was the overflow of an upstream tank containing high BOD whitewater, resulting in spikes of organic acids to the treatment and promoting the proliferation of certain types of filamentous bacteria. A storage response by filamentous bacteria to these high readily biodegradable substrate conditions was monitored with fbATP. By predicting the onset of bulking conditions, this technique can potentially assist operators to make corrective actions proactively.

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture.

Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae-related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (∼ 17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX).

Crystal structure of the ATP-dependent maturation factor of Ni,Fe-containing carbon monoxide dehydrogenases.

CooC proteins are ATPases involved in the incorporation of nickel into the complex active site ([Ni-4Fe-4S]) cluster of Ni,Fe-dependent carbon monoxide dehydrogenases. The genome of the carboxydotrophic bacterium Carboxydothermus hydrogenoformans encodes five carbon monoxide dehydrogenases and three CooC-type proteins, of which CooC1 was shown to be a nickel-binding ATPase. We determined the crystal structure of CooC1 in four different states: empty, ADP-bound, Zn(2+)/ADP-bound, and Zn(2+)-bound. The structure of CooC1 consists of two spatially separated functional modules: an ATPase module containing the deviant Walker A motif and a metal-binding module that confers the specific function of CooC1. The ATPase module is homologous to other members of the MinD family and, in analogy to the dimeric structure of ATP-bound Soj, is likely responsible for the ATP-dependent dimerization of CooC1. Its core topology classifies CooC1 as a member of the MinD family of SIMIBI (signal recognition particle, MinD and BioD)-class NTPases. The crystal structure of Zn(2+)-bound CooC1 reveals a conserved C-X-C motif as the metal-binding site responsible for metal-induced dimerization. The competitive binding of Ni(2+) and Zn(2+) to CooC1 in solution confirms that the conserved C-X-C motif is also responsible for the interaction with Ni(2+). A comparison of the different CooC1 structures determined suggests a mutual dependence of metal-binding site and nucleotide-binding site.

[Non-heme iron proteins as an alternative system of antioxidant defense in the cells of strictly anaerobic microorganisms: a review].

Enzymatic systems accounting for the relative oxygen resistance of multiple strict anaerobes are reviewed, with emphasis on molecular-biological properties and action mechanisms of non-heme iron proteins (neelaredoxins, desulfoferrodoxins, and rubrerythrins). These unique proteins, which are widespread in anaerobes, comprise a system of antioxidant defense against toxic effects of oxygen and products of its incomplete reduction (an alternative to the classic antioxidant system involving superoxide dismutase and catalase). The role of the superoxide reductase-mediated elimination of endogenous superoxide radicals is discussed. This extremely efficient means of rapid superoxide radical detoxification underlies the preferred mechanism for maintaining the optimum balance between oxidized and reduced forms of some proteins in the cells of strict anaerobes.

Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria.

The biogeochemical nitrogen cycle is mediated by many groups of microorganisms that harbour octahaem cytochromes c (OCC). In this study molecular evolutionary analyses and the conservation of predicted functional residues and secondary structure were employed to investigate the descent of OCC proteins related to hydroxylamine oxidoreductase (HAO) and hydrazine oxidoreductase (HZO) from pentahaem cytochrome c nitrite reductase (NrfA). An octahaem cytochrome cnitrite reductase (ONR) was shown to be a possible intermediate in the process. Analysis of genomic neighbourhoods of OCC protein-encoding genes revealed adjacent conserved genes whose products, together with HAO, provide a path of electron transfer to quinone and constitute a functional catabolic module. The latter has evolved more than once under a variety of functional pressures on the catabolic lifestyles of their bacterial hosts. Structurally, the archetypical long helices in the large C-terminal domain of the proteins as well as the distal axial ligands to most haems were highly conserved in NrfA and all descendents. Residues known to be involved in the nitrite reductase activity of NrfA including the 'CxxCK' motif at the catalytic haem, the substrate and Ca binding sites, and the nitrite and ammonium channels were conserved in the eight representatives of ONR. In the latter, a unique cysteine has been inserted above the active site. The 64 other OCC proteins differed from ONR by the absence of the 'CxxCK' motif, the channel residues and most of the Ca-binding residues and the conserved presence of an 'Asp-His' pair inserted above the active site as well as the tyrosine that forms an intersubunit cross-link to the catalytic haem of HAO. Our proposed scenario of evolution of OCC proteins in the HAO family from NrfA is supported by (i) homology based on sequence and structure, (ii) its wide distribution among bacterial taxa, (iii) the dedicated interaction with specific proteins, and it is (iv) congruent with geological history.

Another multiheme protein, hydroxylamine oxidoreductase, abundantly produced in an anammox bacterium besides the hydrazine-oxidizing enzyme.

A hydroxylamine oxidoreductase (HAO) was purified from anammox sludge in which an anammox bacterium, strain KSU-1, was dominant. The enzyme was a 118-kDa homodimer composed of a 53-kDa subunit. With phenazine methosulfate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide as electron acceptors, the V(max) and K(m) for hydroxylamine were determined as 9.6+/-0.2 micromol/min x mg and 33+/-2 microM, while those for hydrazine were 0.54+/-0.0 micromol/min x mg and 25+/-2 microM, respectively. The HAO had a P468 chromophore. These enzymatic properties were different from those of the hydrazine-oxidizing enzyme (HZO), a multiheme protein abundantly produced by the KSU-1 strain, but were similar to those of the HAO purified from Candidatus Brocadia anammoxidans. The hao gene exists upstream of the hzoB gene, which codes for the HZO. The sequence deduced from the hao gene indicated eight c-type heme binding motifs and showed 87% identity with a polypeptide encoded by an open reading frame (kustc1061) in the genome of an anammox bacterium Candidatus Kuenenia stuttgartiensis. These suggested that the HAO is an indispensable enzyme and well conserved in anammox bacteria, similar to the HZO. This enzyme might therefore be a specific hydroxylamine oxidoreductase for anammox bacteria.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of Pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99 percent 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97 percent, 98 percent and 99 percent 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

Avaliou-se a biodiversidade e a importância industrial de bactérias indígenas da mina de sal Khewra, Paquistão. Efetuou-se a amplificação do 16S rDNA dos isolados por PCR empregando-se os iniciadores universais FD1 e rP1, e os produtos foram seqüenciados comercialmente. Essas seqüências de genes foram comparadas com outras seqüências disponíveis no GenBank a fim de encontrar seqüências relacionadas, construindo-se uma árvore filogenética para essas bactérias. Os genes foram depositados no GenBank obtendo-se os números de acesso. A maioria dos isolados pertenceu a diferentes espécies do gênero Bacillus, apresentando 92-99 por cento de identidade de 16S rDNA com a respectiva cepa de referencia. Outros isolados apresentaram alta similaridade com Escherichia coli, Staphylococcus arlettae e Staphylococcus gallinarum, com 97 por cento, 98 por cento e 99 por cento de similaridade de16S rDNA, respectivamente. A capacidade dos isolados produzirem enzimas industriais (amilase, carboximetilcelulase, xilanase, celulase e protease) foi verificada. Todos os isolados foram testados em placas quanto a degradação de amido, carboximetilcelulose, xilana, celulose e caseína. Os isolados BPT-5, 11, 18, 19 e 25 produziram grandes quantidades de enzimas degradadoras de carboidratos e proteínas. Conclui-se que a mina de Sal Khewra apresenta diferentes grupos de bactérias, que são fontes potenciais de enzimas industriais de aplicação comercial.

An O2-inducible rubrerythrin-like protein, rubperoxin, is functional as a H2O2 reductase in an obligatory anaerobe Clostridium acetobutylicum.

Clostridium acetobutylicum, an obligatory anaerobe, is able to grow microoxically with the accumulation of two functionally unknown O2-induced proteins identified by two-dimensional electrophoresis. One was determined to be a novel type rubrerythrin-like protein, named rubperoxin (Rpr) in this study, that conserves one rubredoxin-type Fe(SCys)(4) site per polypeptide in the N-terminus. Recombinant rubperoxin expressed in E. coli purified in its oxidized form is a dimer with optical absorption maxima at 492, 377, and 277nm. Reduced rubperoxin is rapidly and fully oxidized by a half molar ratio of H2O2 per mole protein, and slowly oxidized by t-butyl hydroperoxide and O2. Cell-free extracts from microoxically grown cells efficiently reduce rubperoxin when NAD(P)H is used as the electron donor (preferentially reduced by NADH). These results strongly suggest that rubperoxin is involved in NAD(P)H-dependent H2O2 detoxification in vivo.

Toxic effects of zinc on anaerobic microbiota from Zimapán Reservoir (Mexico).

The toxic effects of heavy metals have been extensively documented in different organisms. Nevertheless, a lack of information exists with regard to this topic in the case of autochthonous microorganism communities. The aim of this study was to evaluate the toxic effects of zinc on the anaerobic microorganisms present in the sediment and anoxic water of Zimapán Reservoir (Mexico), with particular focus on dissimilatory sulphate reducing bacteria. In the laboratory, a system of enrichment microcosms was set up with sediment and water from the reservoir. ATP, protein, carbohydrates and lactate and alcohol dehydrogenase activity were determined. The physicochemical parameters of the reservoir were evaluated over the course of one year. Sulphate reduction occurred in the reservoir throughout the year, but was most pronounced at the end of the wet season and during winter. In the field, increases in the rate of sulphate reduction coincided with the lowest levels of total phosphorus and hydrosoluble organic carbon. Zinc enrichment was observed to modify protein and carbohydrate content as well as to affect lactate and alcohol dehydrogenase activity. All responses followed a zinc concentration-response relationship and were dependent on reservoir physicochemical parameters. ATP content was used as a biomarker to evaluate the sublethal toxic effects of zinc. The acceptable threshold concentration of zinc in the aquatic and sediment enrichment microcosms was determined to be 0.06mgZn/L and 711.1mgZn/kg, respectively.

Isolation of a multiheme protein with features of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizing enrichment culture.

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V(max) and K(m) for hydrazine are 6.2 +/- 0.3 micromol/min.mg and 5.5 +/- 0.6 microM, respectively. Hydrazine (25 microM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 microM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of "Candidatus Kuenenia stuttgartiensis" (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.

[Investigation of the catabolism of acetate and peptides in the new anaerobic thermophilic bacterium Caldithrix abyssi].

This work is concerned with the metabolism of Caldithrix abyssi-an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO2 occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate:ferredoxin oxidoreductase (2.6 micromol/(min mg protein)), phosphate acetyltransferase (0.46 micromol/(min mg protein)), and acetate kinase (0.3 micromol/(min mg protein)). The activity of fumarate reductase (0.14 micromol/(min mg protein)), malate dehydrogenase (0.17 micromol/(min mg protein)), and fumarate hydratase (1.2 micromol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents.

Radical enzymes in anaerobes.

This review describes enzymes that contain radicals and/or catalyze reactions with radical intermediates. Because radicals irreversibly react with dioxygen, most of these enzymes occur in anaerobic bacteria and archaea. Exceptions are the families of coenzyme B(12)- and S-adenosylmethionine (SAM)-dependent radical enzymes, of which some members also occur in aerobes. Especially oxygen-sensitive radical enzymes are the glycyl radical enzymes and 2-hydroxyacyl-CoA dehydratases. The latter are activated by an ATP-dependent one-electron transfer and act via a ketyl radical anion mechanism. Related enzymes are the ATP-dependent benzoyl-CoA reductase and the ATP-independent 4-hydroxybenzoyl-CoA reductase. Ketyl radical anions may also be generated by one-electron oxidation as shown by the flavin-adenine-dinucleotide (FAD)- and [4Fe-4S]-containing 4-hydroxybutyryl-CoA dehydratase. Finally, two radical enzymes are discussed, pyruvate:ferredoxin oxidoreductase and methane-forming methyl-CoM reductase, which catalyze their main reaction in two-electron steps, but subsequent electron transfers proceed via radicals.

rdlA, a new gene encoding a rhodanese-like protein in Halanaerobium congolense and other thiosulfate-reducing anaerobes.

The recently described anaerobic moderately halophilic bacterium Halanaerobium congolense has been shown to reduce thiosulfate and sulfur-but not sulfate-into sulfide. When cultivated in the presence of thiosulfate as terminal electron acceptor, H. congolense possesses a highly active thiosulfate:cyanide sulfur-transferase activity (rhodanese-like enzyme). A gene library of H. congolense (DSM 11287T) was constructed, and a 3.1-kb Sau3A DNA that encompassed a thiosulfate:cyanide sulfur-transferase-encoding gene was isolated in Escherichia coli. This fragment contains 2 orfs, which were separately subcloned in E. coli. The 900-bp gene encoding the rhodanese-like protein was named rdlA. RdlA differs from other known rhodanese-like proteins by having two potential catalytic sites, one N-terminal and one C-terminal, both harboring a cysteine. The two putative active sites are preceded by a highly-conserved region of unknown function. Closely related genes were also characterized in other thiosulfate-reducing non-sulfate-reducing anaerobes belonging to phylogenetically distant microorganisms, thus suggesting that RdlA is of importance in the mechanism of thiosulfate reduction by numerous members of the domain Bacteria.

Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions.

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in 'Birch-like' reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., -1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.