Biotransformations of carboxylated aromatic compounds by the acetogen Clostridium thermoaceticum: generation of growth-supportive CO2 equivalents under CO2-limited conditions.

Clostridium thermoaceticum ATCC 39073 converted vanillate to catechol. Although carboxylated aromatic compounds which did not contain methoxyl groups were not by themselves growth supportive, protocatechuate and p-hydroxybenzoate (nonmethoxylated aromatic compounds) were converted to catechol and phenol, respectively, during carbon monoxide-dependent growth. Syringate is not subject to decarboxylation by C. thermoaceticum (Z. Wu, S. L. Daniel, and H. L. Drake, J. Bacteriol. 170:5705-5708, 1988), and sustained growth at the expense of syringate-derived methoxyl groups was dependent on supplemental CO2. In contrast, vanillate was growth supportive in the absence of supplemental CO2, and 14CO2 was the major 14C-labeled product during [carboxyl-14C]vanillate-dependent growth. Furthermore, the decarboxylation of protocatechuate and p-hydroxybenzoate supported methanol- and 1,2,3-trimethoxybenzene-dependent growth (CO2 is required for growth at the expense of these substrates) when supplemental CO2 was depleted from the growth medium, and the decarboxylation of protocatechuate was concomitant with improved cell yields of methanol cultures. These findings demonstrate that (i) C. thermoaceticum is competent in the decarboxylation of certain aromatic compounds and (ii) under certain conditions, decarboxylation may be integrated to the flow of carbon and energy during acetogenesis.     

CO dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in CO2 and argon atmospheres

The EPR and redox properties of the metal complexes in CO dehydrogenase (CODH) from Clostridium thermoaceticum were studied. Controlled potential coulometric reductive titrations of CODH were performed under argon and CO2 atmospheres. In the titrations performed under argon, five to eight electrons/dimer were required for reduction, and four distinct EPR signals appeared. These included a signal with gave = 1.82 (Em approximately -220 mV), two signals with the same g values but different linewidths at gave = 1.94 (Em approximately -440 mV), and a signal at gave = 1.86 (Em approximately -530 mV). All of the S = 1/2 EPR signals had low spin concentrations; values between 0.2 and 0.3 spins/dimer were typically obtained for each signal. Features between g = 6 and 4, typical of S = 3/2 states, were also observed, and these may account, at least to some degree, for the low spin concentration values. Under CO2, and at negative potentials, CODH served as an electrocatalyst in the reduction of CO2 to CO. The apparent half-maximal activity for this reduction at pH 6.3 occurred at -430 mV, a potential near the thermodynamic value. An EPR signal, arising from a complex containing Ni, Fe, and the carbon from CO/CO2 developed along with this activity. The reduction of this complex is probably the last step to occur prior to the catalysis of CO2 reduction.     

Evidence for a proposed intermediate redox state in the CO/CO(2) active site of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium thermoaceticum

When samples of the enzyme in the C(red1) state were reduced with Ti(3+) citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded C(red2). The EPR-silent state formed within 1 min of adding Ti(3+) citrate, while C(red2) formed after 60 min. Ti(3+) citrate appeared to slow the rate by which C(red2) formed from C(red1) and stabilize the C-cluster in the previously proposed C(int) state. This is the first strong evidence for C(int), and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti(3+) citrate had a different effect on enzyme in a CO(2) atmosphere; it shifted reduction potentials of metal centers (relative to those obtained using CO) and did not stabilize C(int). Different redox behavior was also observed when methyl viologen and benzyl viologen were used as reductants. This variability was exploited to prepare enzyme samples in which EPR from C(red2) was present without interfering signals from B(red). The saturation properties of B(red) depended upon the redox state of the enzyme. Three saturation "modes", called Sat1-Sat3, were observed. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2. 04 and 1.90 resonances, and was observed in samples poised at slightly negative potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO(2). Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was observed at the most negative potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.     

Clostridium mayombei sp. nov., an H2/CO2 acetogenic bacterium from the gut of the African soil-feeding termite,Cubitermes speciosus

Clostridium mayombei sp. nov., a previously undescribed H₂-oxidizing CO₂-reducing acetogenic bacterium, was isolated from gut contents of the African soilfeeding termite, Cubitermes speciosus. Cells were anaerobic, Gram positive, catalase and oxidase negative, endospore-forming motile rods which measured 1×2 – 6 m and which had a DNA base composition of 25.6 mol% G+C (strain SFC-5). Optimum conditions for growth on H₂+CO₂ were at 33°C and pH 7.3, and under these conditions cells produced acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other substrates supporting good growth included carbohydrates (e.g. glucose, xylose, starch), sugar alcohols, and organic and amino acids, and with these substrates acetate was almost always the principle fermentation product. Comparative analysis of 16S rRNA nucleotide sequences confirmed that C. mayombei was closely related to various members of the genus Clostridium. However, morphological and physiological differences between C. mayombei and other homoacetogenic clostridia were deemed significant enough to warrant creation of a new taxon. Results are discussed in light of the diversity of H₂/CO₂ acetogens recently isolated from various termites, and in terms of the relative importance of H₂/CO₂ acetogenesis to termite nutrition.     

The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum)

This paper describes the genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the Wood-Ljungdahl pathway of CO and CO(2) fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using H(2) and CO as electron donors and CO(2) as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO(2). Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols and aromatic compounds) and electron acceptors (CO(2), nitrate, nitrite, thiosulfate, dimethylsulfoxide and aromatic carboxyl groups). The genome consists of a single circular 2 628 784 bp chromosome encoding 2615 open reading frames (ORFs), which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. A total of 2384 (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in orthologue clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.     

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.     

Function and CO binding properties of the NiFe complex in carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Adding 1,10-phenanthroline to carbon monoxide dehydrogenase from Clostridium thermoaceticum results in the complete loss of the NiFeC EPR signal and the CO/acetyl-CoA exchange activity. Other EPR signals characteristic of the enzyme (the gav = 1.94 and gav = 1.86 signals) and the CO oxidation activity are completely unaffected by the 1,10-phenanthroline treatment. This indicates that there are two catalytic sites on the enzyme; the NiFe complex is required for catalyzing the exchange and acetyl-CoA synthase reactions, while some other site is responsible for CO oxidation. The strength of CO binding to the NiFe complex was examined by titrating dithionite-reduced enzyme with CO. During the titration, the NiFeC EPR signal developed to a final spin intensity of 0.23 spin/alpha beta. The resulting CO titration curve (NiFeC spins/alpha beta vs CO pha beta) was fitted using two reactions: binding of CO to the oxidized NiFe complex, and reduction of the CO-bound species to a form that exhibits the NiFeC signal. Best fits yielded apparent binding constants between 6000 and 14,000 M-1 (Kd = 70-165 microM). This sizable range is due to uncertainty whether CO binds to all or only a small fraction (approximately 23%) of the NiFe complexes. Reduction of the CO-bound NiFe complex is apparently required to activate it for catalysis. The electron used for this reduction originates from the CO oxidation site, suggesting that delivery of a low-potential electron to the CO-bound NiFe complex is the physiological function of the CO oxidation reaction catalyzed by this enzyme.     

Adaptation to Low CO(2) Level in a Mutant of Anacystis nidulans R(2) which Requires High CO(2) for Growth

The mutant E₁ of Anacystis nidulans R₂ requires high CO₂ concentration for growth but was able to adapt to low CO₂ concentration. This was exhibited by the increased ability to accumulate inorganic carbon within the cells and the large increase in the amount of a 42-kilodalton polypeptide located in the cytoplasmic membrane. The adaptation occurred in E₁ cells at an extracellular CO₂ concentration as high as 0.3%, which was 8 times the concentration for maximal adaptation in R₂ cells. The ability of E₁ cells to exhibit low CO₂ characteristics at a higher CO₂ concentration was attributed to lower intracellular CO₂ concentration.     

Acetonema longum gen.nov.sp.nov., an H2/CO2 acetogenic bacterium from the termite,Pterotermes occidentis

A previously undescribed, H₂-oxidizing CO₂-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic, Gram-negative, endospore-forming motile rods which measured 0.30–0.40×6–60 m. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G+C in their DNA. Optimum conditions for growth on H₂+CO₂ were at 30–33°C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related to, but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites.     

Characterization of the H2- and CO-dependent chemolithotrophic potentials of the acetogens Clostridium thermoaceticum and Acetogenium kivui.

Strains of Clostridium thermoaceticum were tested for H2- and CO-dependent growth in a defined medium containing metals, minerals, vitamins, cysteine-sulfide, CO2-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditions (the defined medium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively. Product stiochiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2----CH3COOH + 0.1 cell C + 0.3 unrecovered C and 6.8CO----CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced significantly higher acetate concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 values for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the YCO value for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source utilized for growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the Vmax values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 micromoles of H2 oxidized per min mg of protein, respectively; the Vmax values for CO dehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 micromoles of CO oxidized per min per mg of protein, respectively.     

A new H2 / CO2-using acetogenic bacterium from the rumen: Description of Ruminococcus schinkii sp. nov.

Abstract Two strains of H2 / CO2-using acetogenic bacteria were isolated from the rumen of suckling lambs. Both strains displayed a coccobacillar morphology and possessed a Gram-positive type cell wall. Numerous organic substrates, including some O-methylated aromatic compounds, were used heterotrophically. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations a new species, Ruminococcus schinkii sp. nov., is proposed.     

Fermentation of single and mixed substrates by the parent and an acid-tolerant, mutant strain of Clostridium thermoaceticum

Growth of the parent and acid-tolerant mutant strains of Clostridiurn thermoaceticum was examined on a variety of substrates and mixtures of substrates. Nondiauxic growth was noted for both strains on combinations of carbohydrates, organic acids, or a carbohydrate and an organic acid. The mutant strain was able to grow on DL-lactate as sole energy source. The parent strain would not grow on lactate as sole energy source but consumed lactate when presented with a second fermentable substrate. Neither strain would grow on formate as sole energy source, but both consumed formate when presented with a second fermentable substrate.     

Synthesis of (S)- and (R)-3-hydroxy acids using cells or purified (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum and the NADP(H) regeneration system of Clostridium thermoaceticum

A purified and partially characterized novel NADP⁺-dependent oxidoreductase from Clostridium tyrobutyricum DSM 1460 was applied for the preparative reduction of several 3-oxo acids to (S)-3-hydroxy acids. (R)-3-Hydroxybutyrate was prepared by the same enzyme selectively dehydrogenating the S enantiomer of (R,S)-3-hydroxybutyrate. The enantiomeric purity of the (S)- and (R)-3-hydroxy acids was at least 98% enantiomeric excess (e.e). NADPH for reductions and NADP⁺ for dehydrogenations were regenerated by applying artificial mediator accepting pyridine nucleotide oxidoreductases in the form of a crude extract of C. thermoaceticum cells. For NADP⁺ regeneration also the system 2-oxoglutarate/glutamate dehydrogenase was used for comparison. Instead of the purified (S)-3-hydroxycarboxylate oxidoreductase, resting cells of C. tyrobutyricum were also applied for reductions and dehydrogenations with substrate concentrations of 200–400 mM leading to products with e.e. values above 96%.Dedicated to Prof. H.G. Floss on the occasion of his 60th birthday     

Effects of decomposed products from Japanese cedar hydrolyzates on acetic acid fermentation by Clostridium thermocellum and Moorella thermoacetica (C. thermoaceticum)

Japanese cedar (Cryptomeria japonica) was treated with hot-compressed water and as decomposed products, the following compounds were recovered: furfural, 5-hydroxymethyl furfural, levoglucosan, lactic acid, glycolic acid, coniferyl alcohol, coniferyl aldehyde and vanillin. The impacts and fermentability of these compounds were studied on acetic acid fermentation by the co-culture of Clostridium thermocellum and Moorella thermoacetica. It was found that furfural, 5-HMF and lignin-derived products strongly limited acetic acid production by free cells. Importantly, co-immobilized C. thermocellum and M. thermoacetica expressed increased tolerance towards the decomposed products and successfully provided acetic acid corresponding to 93% of the theoretical maximum from Japanese cedar hydrolyzates.