Characterization of Clostridium thermocellum JW20

Clostridium thermocellum JW20 (ATCC 31549), which was isolated from a Louisiana cotton bale, grew on cellulose, cellobiose, and xylooligomers and, after adaptation, on glucose, fructose, and xylose in the pH range of 7.5 to 6.1 with T_opt of 60°C, T_max of 69°C, and T_min of above 28°C. Doubling times during growth on cellulose and cellobiose were 6.5 and 2.5 h, respectively. The G+C content of the DNA was 40 mol% (chemical analysis). Growth on cellulose as substrate was totally inhibited in the presence of more than 125 mM sodium sulfate, 300 mM sodium chloride, 250 mM potassium chloride, 200 mM calcium chloride, 125 mM magnesium chloride, 40 mM lactate, or 250 mM acetate. The ratio of the fermentation products ethanol to acetate plus H₂ decreased when the culture was agitated. Agitation otherwise increased the rate of cellulose degradation in a growing culture but not under nongrowth conditions or with cell-free culture supernatant containing the extracellular cellulase. Shaking lowered the concentration of H₂ in the culture broth and thus minimized inhibition by the H₂ formed. Externally added H₂ caused an increased formation of ethanol during growth on cellulose or cellobiose. However, at an atmospheric pressure as high as 355 kPa (50 lb/in²), H₂ did not cause significant growth inhibition beyond an increasing lag phase (up to 24 h). Several criteria to specifically prove the purity of C. thermocellum cultures were suggested.     

Chemoreception by a cell with age-dependent receptor binding sites

The authors determine the time-dependent ligand current into a spherical cell that is covered with a large number of age-dependent receptors. These receptors can be in either of two states: active (i.e., available for ligand binding) or inactive. An active receptor turns inactive upon binding a ligand, and it can reappear as active at some later time. The transition inactive----active is treated as a probabilistic process. The ligand distribution around the cell is determined analytically in terms of this distribution at the cell surface. A set of nonlinear integral equations is derived for the distribution at the cell surface, which is solved numerically. In this way the time-dependent ligand current into the cell as well as the average active receptor population at the cell surface are determined.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

The anaerobic degradation of 3-chloro-4-hydroxybenzoate in freshwater sediment proceeds via either chlorophenol or hydroxybenzoate to phenol and subsequently to benzoate.

To study the anaerobic degradation of the chimera 3-chloro-4-hydroxybenzoate (3-Cl,4-OHB), anaerobic freshwater sediment samples from the vicinity of Athens, Ga., were adapted for the transformation of 4-hydroxybenzoate (4-OHB), 3-chlorobenzoate (3-CB), 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). In nonadapted samples, both 4-OHB (product of aryl dechlorination) and 2-CP (product of aryl decarboxylation) were observed as intermediates in the transformation of 3-Cl,4-OHB to phenol. The accumulated phenol was subsequently transformed to benzoate, an intermediate in the conversion to methane and CO2. In 4-OHB-adapted samples (i.e., samples adapted for aryl decarboxylation), 2-CP was the first intermediate which was subsequently dechlorinated to phenol. In 3-CB-adapted samples (i.e., samples adapted for meta-chlorobenzoate dehalogenation), 3-Cl,4-OHB was stoichiometrically dechlorinated to 4-OHB. In 2-CP-adapted samples (i.e., samples adapted for ortho-chlorophenol dehalogenation), 4-OHB was the first major intermediate. Furthermore, 3-CB was not dechlorinated in 2-CP-adapted sediment samples, suggesting the possibility that different 3-Cl,4-OHB dechlorinating systems were induced in the 2-CP- and 3-CB-adapted sediments. Adaptation of sediment samples for dechlorination of 2,4-DCP did not lead to adaptation for dechlorination of 3-Cl,4-OHB. However, 3-Cl,4-OHB was dechlorinated to 4-OHB in our stable, sediment-free 2,4-DCP-dechlorinating enrichment, isolated previously from the same environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Reductive dechlorination of dichlorophenols by nonadapted and adapted microbial communities in pond sediments

Fresh and dichlorophenol (DCP)-adapted sediments from two ponds near Athens, Georgia exhibited distinctly different dechlorinating activities. These differences centered on the relative rates of reductive dehlorination in both fresh and adapted sediments and on the substrate specificity of the adapted sediments. Fresh Cherokee Trailer Park Pond sediment dechlorinated 2,3-, 2,4-, and 2,6-DCP to monochlorophenols at a faster rate and after a shorter lag period than fresh Bolton's Pond sediment. Lag periods were not observed in either Cherokee or Bolton's sediments that had been adapted to dechlorinate either 2,3-, 2,4-or 2,6-DCP. Adapted Cherokee sediments exhibited faster dechlorinating rates and a broader substrate specificity than the adapted Bolton's sediments. The broad substrate specificity of each of the adapted Cherokee sediments contrasted sharply with the narrow specificity of the 2,6-DCP-adapted Bolton's sediment. The preference for reductive dechlorination wasortho>meta orpara in sediments from both ponds.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

A High-Molecular-Weight, Cell-Associated Xylanase Isolated from Exponentially Growing Thermoanaerobacterium sp. Strain JW/SL-YS485

An unusual cell-associated (beta)-1,4-xylanase was purified to gel electrophoretic homogeneity from a cell extract of the bacterium Thermoanaerobacterium sp. strain JW/SL-YS485 harvested at the late exponential growth phase. The molecular mass of the xylanase was 350 kDa as determined by gel filtration and 234 kDa as determined by native gradient gel electrophoresis. The enzyme contained 6% carbohydrates. Heterosubunits of 180 and 24 kDa were observed for the xylanase on sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis gels. The xylanase had a pI of 4.37 and a half-life of 1 h at 70(deg)C. Using a 5-min assay, we observed the highest level of activity at pH 6.2 and 80(deg)C. The K(infm) and k(infcat) values when oat spelt xylan was used were 3 mg/ml and 26,680 U/(mu)mol, respectively. The Arrhenius energy was 41.8 kJ/mol. The purified enzyme differed in size, subunit structure, and location from other xylanases that have been described. The cell-associated enzyme activity appeared in the S-layer fraction.