AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation.

Oxidation of phosphite (HPO32-) to phosphate (HPO42-) releases electrons at a very low redox potential (E0'= -690 mV) which renders phosphite an excellent electron donor for microbial energy metabolism. To date, two pure cultures of strictly anaerobic bacteria have been isolated that run their energy metabolism on the basis of phosphite oxidation, the Gram-negative Desulfotignum phosphitoxidans (DSM 13687) and the Gram-positive Phosphitispora fastidiosa (DSM 112739). Here, we describe the key enzyme for dissimilatory phosphite oxidation in these bacteria. The enzyme catalyzed phosphite oxidation in the presence of adenosine monophosphate (AMP) to form adenosine diphosphate (ADP), with concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). The enzyme of P. fastidiosa was heterologously expressed in Escherichia coli. It has a molecular mass of 35.2 kDa and a high affinity for phosphite and NAD+. Its activity was enhanced more than 100-fold by addition of ADP-consuming adenylate kinase (myokinase) to a maximal activity between 30 and 80 mU x mg protein-1. A similar NAD-dependent enzyme oxidizing phosphite to phosphate with concomitant phosphorylation of AMP to ADP is found in D. phosphitoxidans, but this enzyme could not be heterologously expressed. Based on sequence analysis, these phosphite-oxidizing enzymes are related to nucleotide-diphosphate-sugar epimerases and indeed represent AMP-dependent phosphite dehydrogenases (ApdA). A reaction mechanism is proposed for this unusual type of substrate-level phosphorylation reaction.

Anaerobic dissimilatory phosphite oxidation, an extremely efficient concept of microbial electron economy.

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (-690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+ -dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate.

BACKGROUND: Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA. RESULTS: Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate-CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent KM values for isophthalate, CoA, and ATP were 409 µM, 642 µM, and 3580 µM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions. CONCLUSION: We have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate.

Quantification of archaea-driven freshwater nitrification from single cell to ecosystem levels.

Deep oligotrophic lakes sustain large populations of the class Nitrososphaeria (Thaumarchaeota) in their hypolimnion. They are thought to be the key ammonia oxidizers in this habitat, but their impact on N-cycling in lakes has rarely been quantified. We followed this archaeal population in one of Europe's largest lakes, Lake Constance, for two consecutive years using metagenomics and metatranscriptomics combined with stable isotope-based activity measurements. An abundant (8-39% of picoplankton) and transcriptionally active archaeal ecotype dominated the nitrifying community. It represented a freshwater-specific species present in major inland water bodies, for which we propose the name "Candidatus Nitrosopumilus limneticus". Its biomass corresponded to 12% of carbon stored in phytoplankton over the year´s cycle. Ca. N. limneticus populations incorporated significantly more ammonium than most other microorganisms in the hypolimnion and were driving potential ammonia oxidation rates of 6.0 ± 0.9 nmol l‒1 d‒1, corresponding to potential cell-specific rates of 0.21 ± 0.11 fmol cell-1 d-1. At the ecosystem level, this translates to a maximum capacity of archaea-driven nitrification of 1.76 × 109 g N-ammonia per year or 11% of N-biomass produced annually by phytoplankton. We show that ammonia-oxidizing archaea play an equally important role in the nitrogen cycle of deep oligotrophic lakes as their counterparts in marine ecosystems.

Phosphitispora fastidiosa gen. nov. sp. nov., a new dissimilatory phosphite-oxidizing anaerobic bacterium isolated from anaerobic sewage sludge.

A new strictly anaerobic bacterium, strain DYL19T, was enriched and isolated with phosphite as the sole electron donor and CO2 as a single carbon source and electron acceptor from anaerobic sewage sludge sampled at a sewage treatment plant in Constance, Germany. It is a Gram-positive, spore-forming, slightly curved, rod-shaped bacterium which oxidizes phosphite to phosphate while reducing CO2 to biomass and small amounts of acetate. Optimal growth is observed at 30 °C, pH 7.2, with a doubling time of 3 days. Beyond phosphite, no further inorganic or organic electron donor can be used, and no other electron acceptor than CO2 is reduced. Sulphate inhibits growth with phosphite and CO2. The G+C content is 45.95 mol%, and dimethylmenaquinone-7 is the only quinone detectable in the cells. On the basis of 16S rRNA gene sequence analysis and other chemotaxonomic properties, strain DYL19T is described as the type strain of a new genus and species, Phosphitispora fastidiosa gen. nov., sp. nov.

Pelorhabdus rhamnosifermentans gen. nov., sp. nov., a strictly anaerobic rhamnose degrader from freshwater lake sediment.

A rhamnose-degrading bacterium, strain BoRhaAT, was isolated from profundal sediment of Lake Constance in agar dilution series with l-rhamnose as substrate and with a background lawn of Methanospirillum hungatei. The isolated strain was a motile rod that stained Gram positive. Growth was observed within a pH range of 4.0-7.5 and a temperature range of 15-30°C. Fermentation products of rhamnose or glucose were acetate, propionate, ethanol, butyrate, and 1-propanol. The G+C content was 40.6% G+C. The dominant fatty acids are C16:1ω9c, i-C13:03OH, C16:0 and C17:1ω8c with 8-21% relative abundance. Polar lipids were glycolipids, phosphatidylethanolamine, phosphoaminolipid and other lipids, of which phosphatidylethanolamine was most abundant. The sequence of the 16S rRNA gene of the new isolate matches the sequence of its closest relative Anaerosporomusa subterranea to 92.4%. A comparison of the genome with this strain showed 60.2% genome-wide average amino acid identity (AAI), comparisons with other type strains showed a maximum of 62.7% AAI. Thus, the definition of a new genus is justified for which we propose the name Pelorhabdus. For strain BoRhaAT, we propose the name Pelorhabdus rhamnosifermentans gen. nov., sp. nov., with strain BoRhaAT (DSM 111565T = JCM 39158T) as the type strain.

Two Marine Desulfotomaculum spp. of Different Origin are Capable of Utilizing Acetone and Higher Ketones.

Degradation of acetone and higher ketones has been described in detail for aerobic and nitrate-reducing bacteria. Among sulfate-reducing bacteria, degradation of acetone and other ketones is still an uncommon ability and has not been understood completely yet. In the present work, we show that Desulfotomaculum arcticum and Desulfotomaculum geothermicum are able to degrade acetone and butanone. Total proteomics of cell-free extracts of both organisms indicated an involvement of a thiamine diphosphate-dependent enzyme, a B12-dependent mutase, and a specific dehydrogenase during acetone degradation. Similar enzymes were recently described to be involved in acetone degradation by Desulfococcus biacutus. As there are so far only two described sulfate reducers able to degrade acetone, D. arcticum and D. geothermicum represent two further species with this capacity. All these bacteria appear to degrade acetone via the same set of enzymes and therefore via the same pathway.

Activation of short-chain ketones and isopropanol in sulfate-reducing bacteria.

BACKGROUND: Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA. RESULTS: Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B12-dependent mutase, and a NAD+-dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B12-dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B12-dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone. CONCLUSIONS: According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

Desulfatiglans anilini Initiates Degradation of Aniline With the Production of Phenylphosphoamidate and 4-Aminobenzoate as Intermediates Through Synthases and Carboxylases From Different Gene Clusters.

The anaerobic degradation of aniline was studied in the sulfate-reducing bacterium Desulfatiglans anilini. Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes of D. anilini grown with different substrates (aniline, 4-aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grown D. anilini cells but not in phenol- or benzoate-grown cells were located in the putative gene clusters ani (aniline degradation), hcr (4-hydroxybenzoyl-CoA reductase) and phe (phenol degradation). Of these putative gene clusters, only the phe gene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced in E. coli. The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene cluster ani and the other from gene cluster phe, were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn2+, K+, and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacterium D. anilini only by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, that D. anilini carboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate.

Resorcinol Hydroxylase of Azoarcus anaerobius: Molybdenum Dependence, Activity, and Heterologous Expression.

The obligately anaerobic, denitrifying bacterium Azoarcus anaerobius strain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe-4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed in Escherichia coli but the products remained in inclusion bodies. All attempts to purify RH from A. anaerobius or to produce soluble, active RH in E. coli failed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 in Thauera aromatica AR1 transconjugants carrying a transposon insertion in the coding gene for the large (ΔrhL) or the small subunit (ΔrhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugant T. aromatica AR1/R+ showed a specific activity of 80 mU mg-1, and the specific activity of RH in the membranes of the complemented mutants was in the same range (80-95 mU mg-1). We conclude that RH of A. anaerobius is a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.

Desulfolutivibrio sulfoxidireducens gen. nov., sp. nov., isolated from a pyrite-forming enrichment culture and reclassification of Desulfovibrio sulfodismutans as Desulfolutivibrio sulfodismutans comb. nov.

Two strains of sulfate-reducing bacteria (J.5.4.2-L4.2.8T and J.3.6.1-H7) were isolated from a pyrite-forming enrichment culture and were compared phylogenetically and physiologically to the closest related type strain Desulfovibrio sulfodismutans DSM 3696T. The isolated strains were vibrio-shaped, motile rods that stained Gram-negative. Growth occurred from 15 to 37°C and within a pH range of 6.5-8.5. Both strains used sulfate, thiosulfate, sulfite, and dimethyl sulfoxide (DMSO) as electron acceptor when grown with lactate. Lactate was incompletely oxidized to acetate. Formate and H2 were used as electron donor in the presence of acetate. Dismutation of thiosulfate and pyrosulfite was observed. The two new isolates differed from D. sulfodismutans by the utilization of DMSO as electron acceptor, 82% genome-wide average nucleotide identity (ANI) and 32% digital DNA-DNA hybridization (dDDH), thus representing a novel species. The type strain of the type species Desulfovibrio desulfuricans Essex6T revealed merely 88% 16S rRNA gene identity and 49% genome-wide average amino acid identity (AAI) to the new isolates as well as to D. sulfodismutans. Furthermore, the dominance of menaquinone MK-7 over MK-6 and the dominance of ai-C15:0 fatty acids were observed not only in the two new isolated strains but also in D. sulfodismutans. Therefore, the definition of a new genus is indicated for which the name Desulfolutivibrio is proposed. We propose for strains J.5.4.2-L4.2.8T and J.3.6.1-H7 the name Desulfolutivibrio sulfoxidireducens gen. nov. sp. nov. with strain J.5.4.2-L4.2.8T defined as type strain. In addition, we propose the reclassification of Desulfovibrio sulfodismutans as Desulfolutivibrio sulfodismutans comb. nov.

Further guidelines for the formation of compound specific and subspecific epithets. A proposal to emend Appendix 9 of the International Code of Nomenclature of Prokaryotes.

We propose emending section A(1)(b) of Appendix 9 of the International Code of Nomenclature of Prokaryotes with further guidelines for the formation of compound specific or subspecific epithets based on localities and epithets based on binomial names of plants or animals.

Use of Greek in the prokaryotic nomenclature: proposal to change Principle 3, Recommendation 6, Rule 7, Rule 65 and Appendix 9 of the International Code of Nomenclature of Prokaryotes.

Principle 3 of the International Code of Nomenclature of Prokaryotes (ICNP) states that the scientific names of all taxa are Latin or latinized words treated as Latin regardless of their origin. They are usually taken from Latin or Greek. Recently we encountered cases where newly proposed names were based on words from Modern Greek that are not derived from words found in the dictionaries of Classical Greek. In our opinion, there is no special reason why Modern Greek words not found in the classical language should have a special status in the ICNP. We therefore propose modifying Principle 3, Recommendation 6, Rule 7, Rule 65 and Appendix 9 of the ICNP to specify the special status of Classical Greek besides Latin.

Physiological limits to life in anoxic subseafloor sediment.

In subseafloor sediment, microbial cell densities exponentially decrease with depth into the fermentation zone. Here, we address the classical question of 'why are cells dying faster than they are growing?' from the standpoint of physiology. The stoichiometries of fermentative ATP production and consumption in the fermentation zone place bounds on the conversion of old cell biomass into new. Most fermentable organic matter in deep subseafloor sediment is amino acids from dead cells because cells are mostly protein by weight. Conversion of carbon from fermented dead cell protein into methanogen protein via hydrogenotrophic and acetoclastic methanogenesis occurs at ratios of ∼200:1 and 100:1, respectively, while fermenters can reach conversion ratios approaching 6:1. Amino acid fermentations become thermodynamically more efficient at lower substrate and product concentrations, but the conversion of carbon from dead cell protein into fermenter protein is low because of the high energetic cost of translation. Low carbon conversion factors within subseafloor anaerobic feeding chains account for exponential declines in cellular biomass in the fermentation zone of anoxic sediments. Our analysis points to the existence of a life-death transition zone in which the last biologically catalyzed life processes are replaced with purely chemical reactions no longer coupled to life.

Microbial degradation of phthalates: biochemistry and environmental implications.

The environmentally relevant xenobiotic esters of phthalic acid (PA), isophthalic acid (IPA) and terephthalic acid (TPA) are produced on a million ton scale annually and are predominantly used as plastic polymers or plasticizers. Degradation by microorganisms is considered as the most effective means of their elimination from the environment and proceeds via hydrolysis to the corresponding PA isomers and alcohols under oxic and anoxic conditions. Further degradation of PA, IPA and TPA differs fundamentally between anaerobic and aerobic microorganisms. The latter introduce hydroxyl functionalities by dioxygenases to facilitate subsequent decarboxylation by either aromatizing dehydrogenases or cofactor-free decarboxylases. In contrast, anaerobic bacteria activate the PA isomers to the respective thioesters using CoA ligases or CoA transferases followed by decarboxylation to the central intermediate benzoyl-CoA. Decarboxylases acting on the three PA CoA thioesters belong to the UbiD enzyme family that harbour a prenylated flavin mononucleotide (FMN) cofactor to achieve the mechanistically challenging decarboxylation. Capture of the extremely instable PA-CoA intermediate is accomplished by a massive overproduction of phthaloyl-CoA decarboxylase and a balanced production of PA-CoA forming/decarboxylating enzymes. The strategy of anaerobic phthalate degradation probably represents a snapshot of an ongoing evolution of a xenobiotic degradation pathway via a short-lived reaction intermediate.

Energy-Conserving Enzyme Systems Active During Syntrophic Acetate Oxidation in the Thermophilic Bacterium Thermacetogenium phaeum.

The thermophilic acetogen Thermacetogenium phaeum uses the Wood-Ljungdahl pathway (WLP) in both directions, either for the production of acetate from various compounds or for the oxidation of acetate in syntrophic cooperation with methanogens. In this study, energy-conserving enzyme systems in T. phaeum were investigated in both metabolic directions. A gene cluster containing a membrane-bound periplasmically oriented formate dehydrogenase directly adjacent to putative menaquinone synthesis genes was identified in the genome. The protein products of these genes were identified by total proteome analysis, and menaquinone MK-7 had been found earlier as the dominant quinone in the membrane. Enzyme assays with membrane preparations and anthraquinone-2,6-disulfonate as electron acceptor verified the presence of a quinone-dependent formate dehydrogenase. A quinone-dependent methylene-THF reductase is active in the soluble fraction and in the membrane fraction. From these results we conclude a reversed electron transport system from methyl-THF oxidation to CO2 reduction yielding formate as reduced product which is transferred to the methanogenic partner. The redox potential difference between methyl-THF (Eo' = -200 mV) and formate (Eo' = -432 mV) does not allow electron transfer through syntrophic formate removal alone. We postulate that part of the ATP conserved by substrate-level phosphorylation has to be invested into the generation of a transmembrane proton gradient by ATPase. This proton gradient could drive the endergonic oxidation of methyl-THF in an enzyme reaction similar to the membrane-bound reversed electron transport system previously observed in the syntrophically butyrate-oxidizing bacterium Syntrophomonas wolfei. To balance the overall ATP budget in acetate oxidation, we postulate that acetate is activated through an ATP-independent path via aldehyde:ferredoxin oxidoreductase (AOR) and subsequent oxidation of acetaldehyde to acetyl-CoA.

Enzymes involved in phthalate degradation in sulphate-reducing bacteria.

The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initiated by hydrolysis to alcohols and o-phthalate (phthalate) by esterases. While further catabolism of phthalate has been studied in aerobic and denitrifying microorganisms, the degradation in obligately anaerobic bacteria has remained obscure. Here, we demonstrate a previously overseen growth of the δ-proteobacterium Desulfosarcina cetonica with phthalate/sulphate as only carbon and energy sources. Differential proteome and CoA ester pool analyses together with in vitro enzyme assays identified the genes, enzymes and metabolites involved in phthalate uptake and degradation in D. cetonica. Phthalate is initially activated to the short-lived phthaloyl-CoA by an ATP-dependent phthalate CoA ligase (PCL) followed by decarboxylation to the central intermediate benzoyl-CoA by an UbiD-like phthaloyl-CoA decarboxylase (PCD) containing a prenylated flavin cofactor. Genome/metagenome analyses predicted phthalate degradation capacity also in the sulphate-reducing Desulfobacula toluolica, strain NaphS2, and other δ-proteobacteria. Our results suggest that phthalate degradation proceeds in all anaerobic bacteria via the labile phthaloyl-CoA that is captured and decarboxylated by highly abundant PCDs. In contrast, two alternative strategies have been established for the formation of phthaloyl-CoA, the possibly most unstable CoA ester in biology.

Alternative Pathways of Acetogenic Ethanol and Methanol Degradation in the Thermophilic Anaerobe Thermacetogenium phaeum.

Growth of the anaerobic thermophile Thermacetogenium phaeum with methanol, ethanol, ethanolamine, and acetate was investigated in axenic cultures and in syntrophic cultures with Methanothermobacter thermautotrophicus. Microcompartment genes were identified in the T. phaeum genome, and presence of microcompartments was confirmed by transmission electron microscopy and proteome analysis. These genes were expressed only during growth with ethanolamine. Proteome data were compared after growth with all four substrates, and activities of key enzymes of the Wood-Ljungdahl pathway and of enzyme systems leading to production or degradation of acetaldehyde such as alcohol dehydrogenase, aldehyde:ferredoxin oxidoreductase, acetate kinase, and phosphate acetyltransferase were measured in cytoplasmic fractions. Accounting of fermentation stoichiometries and growth yields with all four substrates showed that ethanol and methanol oxidation follow the same stoichiometries as in Acetobacterium woodii. On the other hand, the pathways of ethanol and methanol degradations vary between both organisms. Growth yields of T. phaeum were substantially lower than reported for A. woodii. Since T. phaeum has no Rnf complex encoded in its genome, the mechanisms of ATP synthesis have to be different from those of A. woodii. In addition to the central degradation pathways also found in A. woodii, T. phaeum maintains enzyme systems that compensate for the absence of an Rnf-complex but which on the other hand cause a loss of energy. On the basis of our data, pathways of methanol and ethanol degradation in T. phaeum are discussed.