Lars G. Ljungdahl

Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new, extreme thermophilic, anaerobic bacterium

Two strains, JW 200 and JW 201, of an extreme thermophilic, non-spore-forming anaerobic bacterium were isolated from alkaline and slightly acidic hot springs located in Yellowstone National Park. Both strains were peritrichously flagellated rods. Cell size varied from 0.5–0.8 by 4–100 μm; coccoid-shaped cells of about 1 μm in diameter frequently occurred. Division was often unequal. Spheroplast-like forms were visible at the late logarithmic growth phase. The Gram reaction was variable. The DNA base composition of the two strains was between 37 and 39 mol% guanine plus cytosine as determined by buoyant density measurements and approximately 32% by the thermal denaturation method. The main fermentation products from hexoses were ethanol and CO2. Growth occurred between 37 and 78°C and from pH 4.4 to 9.8. The name Thermoanaerobacter ethanolicus gen. nov., spec. nov. was proposed for the two, new isolates. Strain JW 200 was designated as the type strain.

Ecology of Microbial Cellulose Degradation

Worldwide photosynthetic fixation of carbon dioxide is estimated to yield annually up to 150 X 109 tons of dry plant material (biomass) (Lieth, 1973; Whittaker and Likens, 1973; Bassham, 1975; Stephens and Heichel, 1975). Almost half of this material consists of cellulose (28–50%); other major components are hemicelluloses (20–30%) and lignin (18–30%) (Thompson, 1983). Additional important but minor constituents of biomass are proteins, lipids, and carbohydrates such as chitin, starch, and pectin. A list of the amounts of cellulose in various plants has been compiled by Stephens and Heichel (1975). The biomass is eventually degraded and oxidized to CO2 and returned to the atmosphere. Thus, we have a carbon cycle. The gain of the cycling process is the capture of solar energy, which is then used by living cells for growth and maintenance.

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.

Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation to plant cell wall degradation

SummaryTrans-feruloyl and trans-p-coumaroyl esterases were found in the culture filtrates of two monocentric (Piromyces MC-1, Neocallimastix MC-2) and three polycentric (Orpinomyces PC-2, Orpinomyces PC-3, and PC-1, an unnamed genus with uniflagellated zoospores) isolates of anaerobic rumen fungi. Treatment of cell walls of Coastal bermudagrass shoots with the filtrates released the trans isomers of ferulic and p-coumaric acids; results of microscopic observations indicated that fungal isolates degraded primarily unlignified cell walls in leaf blades and stems. A greater proportion of ferulic than p-coumaric acid was released by this treatment when compared with the amounts of the acids released by saponification of the walls with 1 M NaOH. The filtrates also showed esterase activities against the trans isomers of methyl ferulate and methyl p-coumarate, with ferulic acid being released at a faster rate than p-coumaric acid. Assays for other cell-wall-degrading enzymes (xylanase, β-xylosidase, α-l-arabinosidase, cellulase, β-glucosidase) indicated that only β-xylosidase correlated with ferulate and p-coumarate esterase activities. The monocentric isolate MC-2 had the highest esterase activity against both the plant cell wall and methyl ester substrates and the highest specific activities of acetyl esterase, β-xylosidase, α-l-arabinosidase, cellulase and β-glucosidase. Isolate MC-2 produced substantially greater amounts of feruloyl and p-coumaroyl esterase when the growth substrate contained higher levels of saponifiable ferulic and p-coumaric acids.

Feruloyl Esterase Activity of the Clostridium thermocellum Cellulosome Can Be Attributed to Previously Unknown Domains of XynY and XynZ

The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, beta-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungus Orpinomyces. Purified cellulosomes from C. thermocellum were found to hydrolyze FAXX (O-(5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D- xyl opyranosyl-(1-->4)-D-xylopyranose) and FAX(3) (5-O-[(E)-feruloyl]-[O-beta-D-xylopyranosyl-(1-->2)]-O-alpha-L- arabinofuranosyl-[1-->3])-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose) , yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia coli, and the expressed proteins hydrolyzed FAXX and FAX(3). The recombinant feruloyl esterase domain of XynZ alone (FAE(XynZ)) and with the adjacent cellulose binding domain (FAE-CBD(XynZ)) were characterized. FAE-CBD(XynZ) had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. K(m) and V(max) values for FAX(3) were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60 degrees C. PAX(3), a substrate similar to FAX(3) but with a p-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70 degrees C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount of p-coumaric acid. FAE(XynZ) had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAE(XynZ) resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the C. thermocellum cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.

The Fibronectin Type 3-Like Repeat from the Clostridium thermocellum Cellobiohydrolase CbhA Promotes Hydrolysis of Cellulose by Modifying Its Surface

ABSTRACT Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of Clostridium thermocellum are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn31,2), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn31,2 (more efficient), and Gh9-Fn31,2-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn31,2, and then Gh9-Fn31,2-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn31,2, or Fn31,2-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn31,2, Fn31,2-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn31,2 and Fn31,2-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 μmoles g−1 and relative affinities (Kr) of 1.12 and 2.13 liters g−1, respectively. Fn31,2 bound weakly to both celluloses. Fn31,2-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 μmoles g−1 and Krs of 1.14 and 1.98 liters g−1, respectively. Fn31,2 and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn31,2 aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn31,2 on the cellulose surface.

The Cellulase/Hemicellulase System of the Anaerobic FungusOrpinomycesPC‐2 and Aspects of Its Applied Use

Anaerobic fungi, first described in 1975 by Orpin, live in close contact with bacteria and other microorganisms in the rumen and caecum of herbivorous animals, where they digest ingested plant food. Seventeen distinct anaerobic fungi belonging to five different genera have been described. They have been found in at least 50 different herbivorous animals. Anaerobic fungi do not possess mitochondria, but instead have hydrogenosomes, which form hydrogen and carbon dioxide from pyruvate and malate during fermentation of carbohydrates. In addition, they are very oxygen‐ and temperature‐sensitive, and their DNA has an unusually high AT content of from 72 to 87 mol%. My initial reason for studying anaerobic fungi was because they solubilize lignocellulose and produce all enzymes needed to efficiently hydrolyze cellulose and hemicelluloses. Although some of these enzymes are found free in the medium, most of them are associated with cellulosomal and polycellulosomal complexes, in which the enzymes are attached through fungal dockerins to scaffolding proteins; this is similar to what has been found for cellulosomes from anaerobic bacteria. Although cellulosomes from anaerobic fungi share many properties with cellulosomes of anaerobic cellulolytic bacteria and have comparable structures, their structures differ in their amino acid sequences. I discuss some features of the cellulosome of the anaerobic fungus Orpinomyces sp. PC‐2 and some possible uses of its enzymes in industrial settings.

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls☆

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.

[39] Formate dehydrogenase, a selenium-tungsten enzyme from Clostridium thermoaceticum

Publisher Summary This chapter describes the purification procedure of formate dehydrogenase, a selenium–tungsten enzyme from Clostridium thermoaceticum. Formate dehydrogenase of C. thermoaceticum catalyzes the reversible reaction. C. thermoaceticum is a thermophilic anaerobic bacterium that ferments sugars to acetate as the only product. It is a strictly anaerobic bacterium, and it fails to grow in the presence of small amounts of oxygen. The only known naturally occurring electron acceptor is NADP, and the enzyme should perhaps be named formate: NADP oxidoreductase. The physiological role of the enzyme is to catalyze the reduction of CO 2 to formate. C. thermoaceticum (DSM 521) is maintained in agar-stab cultures or in liquid broth cultures. The buffer used in the purification of the formate dehydrogenase from C. thermoaceticum contains thioglycolate and iron. These substrates interfere with the assay of protein if the biuret method or the method by Lowry et al . is used. Therefore, protein is assayed by precipitating with trichloroacetic acid and by determining the turbidity after 10 minutes at 400 nm.

Cytochrome bd Oxidase, Oxidative Stress, and Dioxygen Tolerance of the Strictly Anaerobic Bacterium Moorella thermoacetica

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (approximately 70%) inhibited both the endogenous and CO/methanol-dependent O2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-beta-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b561, cytochrome b595, and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine + H2S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.